Anti-GITR Antibodies and Methods of Use Thereof

ABSTRACT

The present disclosure provides antibodies that specifically bind to human glucocorticoid-induced TNFR family related receptor (GITR) and compositions comprising such antibodies. In a specific aspect, the antibodies specifically bind to human GITR and modulate GITR activity, e.g., enhance, activate or induce GITR activity, utilizing such antibodies. The present disclosure also provides methods for treating disorders, such as cancer and infectious diseases, by administering an antibody that specifically binds to human GITR and modulates GITR activity e.g., enhances, activates or induces GITR activity.

REFERENCE TO RELATED APPLICATIONS

Related applications U.S. Provisional Application No. 62/004,071, filedMay 28, 2014, and U.S. Provisional Application No. 62/161,250, filed May13, 2015, are herein incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a sequence listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 28, 2015, isnamed 3617.0090002_SL_PatentIn_ST25.txt and is 747,129 bytes in size.

1. FIELD

The present disclosure provides antibodies that specifically bind tohuman glucocorticoid-induced TNFR family related receptor (GITR) andcompositions comprising such antibodies. In a specific aspect, theantibodies specifically bind to human GITR and modulate GITR activity,e.g., enhance, activate or induce GITR activity, utilizing suchantibodies. The present disclosure also provides methods for treatingdisorders, such as cancer and infectious diseases, by administering anantibody that specifically binds to human GITR and modulates GITRactivity, e.g., enhances, activates or induces GITR activity.

2. BACKGROUND

Glucocorticoid-induced TNFR-related protein (GITR), a member of the TNFRsuperfamily, is expressed in many components of the innate and adaptiveimmune system and stimulates both acquired and innate immunity(Nocentini G et al., (1994) PNAS 94: 6216-6221; Hanabuchi S et al.,(2006) Blood 107:3617-3623; Nocentini G & Riccardi C (2005) Eur JImmunol 35: 1016-1022; Nocentini G et al., (2007) Eur J Immunol37:1165-1169). It is expressed in several cells and tissues, includingT, B, dendritic (DC) and Natural Killer (NK) cells and is activated byits ligand, GITRL, mainly expressed on Antigen Presenting Cells (APCs),on endothelial cells, and also in tumor cells. The GITR/GITRL systemparticipates in the development of autoimmune/inflammatory responses andpotentiates response to infection and tumors. For example, treatinganimals with GITR-Fc fusion protein ameliorates autoimmune/inflammatorydiseases while GITR triggering is effective in treating viral,bacterial, and parasitic infections, as well in boosting immune responseagainst tumors (Nocentini G et al., (2012) Br J Pharmacol 165: 2089-99).These effects are due to several concurrent mechanisms including:co-activation of effector T-cells, inhibition of regulatory T (Treg)cells, NK-cell co-activation, activation of macrophages, modulation ofdendritic cell function and regulation of the extravasation process. Themembrane expression of GITR is increased following T cell activation(Hanabuchi S et al., (2006) supra; Nocentini G & Riccardi C supra). Itstriggering coactivates effector T lymphocytes (McHugh R S et al., (2002)Immunity 16: 311-323; Shimizu J et al., (2002) Nat Immunol 3: 135-142;Roncheti S et al., (2004) Eur J Immunol 34: 613-622; Tone M et al.,(2003) PNAS 100: 15059-15064). GITR activation increases resistance totumors and viral infections, is involved in autoimmune/inflammatoryprocesses and regulates leukocyte extravasation (Nocentini G & RiccardiC (2005) supra; Cuzzocrea S et al., (2004) J Leukoc Biol 76: 933-940;Shevach E M & Stephens G L (2006) Nat Rev Immunol 6: 613-618; CuzzocreaS et al., (2006) J Immunol 177: 631-641; Cuzzocrea S et al., (2007)FASEB J 21: 117-129).

Human GITR is expressed at very low levels in peripheral (non-activated)T cells. After T cell activation, GITR is strongly up-regulated forseveral days in both CD4⁺ and CD8⁺ cells (Kwon B et al., (1999) J BiolChem 274: 6056-6061; Gurney A L et al., (1999) Curr Biol 9: 215-218;Ronchetti S et al., (2004) supra; Shimizu J et al., (2002) supra; Ji H Bet al., (2004) supra; Ronchetti S et al., (2002) Blood 100: 350-352; LiZ et al., (2003) J Autoimmun 21: 83-92), with CD4⁺ cells having a higherGITR expression than CD8⁺ cells (Kober J et al., (2008) Eur J Immunol38(10): 2678-88; Bianchini R et al., (2011) Eur J Immunol 41(8):2269-78).

Given the role of human GITR in modulating immune responses, providedherein are antibodies that specifically bind to GITR and the use ofthose antibodies to modulate GITR activity.

3. SUMMARY

In one aspect, provided herein are antibodies and fragments thereof thatspecifically bind to GITR (e.g., human GITR). In one embodiment, anantibody or antigen-binding fragment thereof that specifically binds toGITR (e.g., human GITR) partially inhibits GITR ligand (e.g., humanGITRL) from binding to GITR as assessed by a method known to one ofskill in the art or described herein (see, e.g., Sections 6.2.5.2 and6.2.5.4, infra). In a specific embodiment, the antibody orantigen-binding fragment thereof at a concentration of 1000 ng/mlinhibits less than 80% of 0.5 nM GITRL (e.g., human GITRL) from bindingto GITR coupled to beads (e.g., human GITR coupled to Luminex® beads) ata concentration of 5 pg/ml/bead relative to the binding of 0.5 nM GITRLto the GITR coupled beads at a concentration of 5 pg/ml/bead in theabsence of the anti-GITR antibody or antigen-binding fragment thereof ina suspension array assay. In certain embodiments, the antibody orantigen-binding fragment thereof inhibits 40% to 70%, 50% to 70%, 50% to80%, or 40% to 80% of the GITRL (e.g., human GITRL) from binding to GITR(e.g., human GITR). In another specific embodiment, at least 20% of theamount of GITRL (e.g., human GITRL) that binds to GITR (e.g., humanGITR) in the absence of the antibody or antigen-binding fragment thereofbinds to GITR (e.g., human GITR) in the presence of the antibody orantigen-binding fragment thereof in an assay: (a) coupling GITR (e.g.,human GITR) to beads at a concentration of 5 pg/ml/bead; (b) incubatingthe GITR (e.g., human GITR) coupled beads at a concentration of 40beads/μl with or without the antibody in a well; (c) adding labeledGITRL (e.g., labeled human GITRL) to the well to obtain a finalconcentration of 0.5 nM of the GITRL (e.g., human GITRL) and 20 beads/μlof the GITR coupled beads; and (d) detecting the labeled GITRL (e.g.,human GITRL) bound to the GITR (e.g., human GITR) coupled beads by,e.g., a suspension array assay. In some embodiments, 20% to 60%, 20% to50%, 30% to 60% or 30% to 50% of the amount of GITRL (e.g., human GITRL)that binds to GITR (e.g., human GITR) in the absence of the antibody orantigen-binding fragment thereof binds to GITR (e.g., human GITR) in thepresence of the antibody or antigen-binding fragment thereof.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises:

(a) a heavy chain variable region (VH) complementarity determiningregion (CDR) 1 comprising, consisting of, or consisting essentially ofthe amino acid sequence of X₁YX₂MX₃ (SEQ ID NO: 1), wherein

X₁ is D, E, G or A;

X₂ is A, V, L, I, P, F, M or Y; and

X₃ is Y, G, N, Q, S, T, C, W, F or H;

(b) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence of X₁IX₂X₃X₄SGX₅X₆X₇YX₈QKFX₉X₁₀ (SEQ ID NO: 2),wherein

X₁ is V, A, L, I, P, F, M or T;

X₂ is R, K, H, Q or A;

X₃ is T, G, N, Q, S, C, W, Y, V, I or P;

X₄ is Y, G, N, Q, S, T, C, W, F, H, or A;

X₅ is D, E, G or A;

X₆ is V, A, L, I, P, F, M or T;

X₇ is T, G, N, Q, S, C, W, Y, V, I, P or A;

X₈ is N, G, Q, S, T, C, W, Y or A;

X₉ is K, R, H, Q or A; and

X₁₀ is D, E, G or A;

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence of SGTVRGX₁X₂X₃ (SEQ ID NO: 3), wherein

X₁ is F, A, V, L, I, P, M, Y, W, H or S;

X₂ is A or D; and

X₃ is Y, G, N, Q, S, T, C, W, F, H or V;

(d) a light chain variable region (VL) CDR1 comprising, consisting of,or consisting essentially of the amino acid sequence ofKSSQSX₁X₂X₃X₄X₅X₆X₇KX₈YLX₉ (SEQ ID NO: 4), wherein:

X₁ is L, A, V, I, P, F or M;

X₂ is L, A, V, I, P, F, M or S;

X₃ is N, G, Q, S, T, C, W, Y or A;

X₄ is S, G, N, Q, T, C, W, Y or A;

X₅ is G, N, Q, S, T, C, W, Y or A;

X₆ is N, G, Q, S, T, C, W, Y or A;

X₇ is Q, G, N, S, T, C, W, Y or A;

X₈ is N, G, Q, S, T, C, W, Y or A; and

X₉ is T, G, N, Q, S, C, W, Y, V, I or A;

(e) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence of X₁ASTRX₂X₃(SEQ ID NO: 5), wherein:

X₁ is W, G, N, Q, S, T, C, Y, F, H or A;

X₂ is E, D or A; and

X₃ is S, G, N, Q, T, C, W, Y or A; and

(f) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence of QX₁X₂YX₃X₄PYT (SEQ ID NO: 6), wherein:

X₁ is N, G, Q, S, T, C, W or Y;

X₂ is D, E or Y; and

X₃ is S, G, N, Q, T, C, W, Y or A, and

X4 is Y, G, N, Q, S, T, C, W, F, H, L, or A.

In other embodiments, the antibody or antigen-binding fragment thereofcomprises:

(a) a heavy chain variable region (VH) CDR1 comprising, consisting of,or consisting essentially of the amino acid sequence of X₁YX₂MX₃ (SEQ IDNO: 7), wherein

X₁ is D, E or G;

X₂ is A or V; and

X₃ is Y or H;

(b) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence of X₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈ (SEQ ID NO: 8),wherein

X₁ is V or L;

X₂ is R, K or Q;

X₃ is Y or F;

X₄ is D, E or G;

X₅ is V or L;

X₆ is T or S;

X₇ is K, R or Q; and

X₈ is D, E or G;

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence of SGTVRGFAY (SEQ ID NO: 9);

(d) a light chain variable region (VL) CDR1 comprising, consisting of,or consisting essentially of the amino acid sequence ofKSSQSLLNSX₁NQKNYLX₂ (SEQ ID NO: 10), wherein

X₁ is G or S; and

X₂ is T or S;

(e) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence of WASTRES (SEQ ID NO: 11); and

(f) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence of QNX₁YSX₂PYT (SEQ ID NO: 12), wherein

X₁ is D or E; and

X₂ is Y, F or S.

In some embodiments, provided herein is an antibody or antigen-bindingfragment thereof that specifically binds to GITR (e.g., human GITR),comprising:

(a) a heavy chain variable region (VH) complementarity determiningregion (CDR) 1 comprising, consisting of, or consisting essentially ofthe amino acid sequence of X₁YX₂MX₃ (SEQ ID NO: 1), wherein

X₁ is D, E, G or A;

X₂ is A, V, L, I, P, F, M or Y; and

X₃ is Y, G, N, Q, S, T, C, W, F or H;

(b) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence of X₁IX₂X₃X₄SGX₅X₆X₇YX₈QKFX₉X₁₀ (SEQ ID NO: 2),wherein

X₁ is V, A, L, I, P, F, M or T;

X₂ is R, K, H, Q or A;

X₃ is T, G, N, Q, S, C, W, Y, V, I or P;

X₄ is Y, G, N, Q, S, T, C, W, F, H, or A;

X₅ is D, E, G or A;

X₆ is V, A, L, I, P, F, M or T;

X₇ is T, G, N, Q, S, C, W, Y, V, I, P or A;

X₈ is N, G, Q, S, T, C, W, Y or A;

X₉ is K, R, H, Q or A; and

X₁₀ is D, E, G or A;

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence of SGTVRGX₁X₂X₃ (SEQ ID NO: 3), wherein

X₁ is F, A, V, L, I, P, M, Y, W, H or S;

X₂ is A, or D; and

X₃ is Y, G, N, Q, S, T, C, W, F, H or V;

(d) a light chain variable region (VL) CDR1 comprising, consisting of,or consisting essentially of the amino acid sequence ofKSSQSX₁X₂X₃X₄X₅X₆X₇KX₈YLX₉ (SEQ ID NO: 4), wherein:

X₁ is L, A, V, I, P, F or M;

X₂ is L, A, V, I, P, F, M or S;

X₃ is N, G, Q, S, T, C, W, Y or A;

X₄ is S, G, N, Q, T, C, W, Y or A;

X₅ is G, N, Q, S, T, C, W, Y or A;

X₆ is N, G, Q, S, T, C, W, Y or A;

X₇ is Q, G, N, S, T, C, W, Y or A;

X₈ is N, G, Q, S, T, C, W, Y or A; and

X₉ is T, G, N, Q, S, C, W, Y, V, I or A;

(e) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence of X₁ASTRX₂X₃(SEQ ID NO: 5), wherein:

X₁ is W, G, N, Q, S, T, C, Y, F, H or A;

X₂ is E, D or A; and

X₃ is S, G, N, Q, T, C, W, Y or A; and

(f) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence of QX₁X₂YX₃X₄PYT (SEQ ID NO: 6), wherein:

X₁ is N, G, Q, S, T, C, W or Y;

X₂ is D, E or Y; and

X₃ is S, G, N, Q, T, C, W, Y or A, and

X4 is Y, G, N, Q, S, T, C, W, F, H, L, or A. In certain embodiments, theantibody or antigen-binding fragment thereof comprises a VH CDR1comprising, consisting of, or consisting essentially of an amino acidsequence selected from the group consisting of SEQ ID NO: 13, 19-23, and117-119. In certain embodiments, the antibody or antigen-bindingfragment thereof comprises a VH CDR1 comprising, consisting of, orconsisting essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 35 and SEQ ID NO:116. In some embodiments, theantibody or antigen-binding fragment thereof comprises a VH CDR2comprising, consisting of, or consisting essentially of an amino acidsequence selected from the group consisting of SEQ ID NO: 14, 24-33, and120-188. In some embodiments, the antibody or antigen-binding fragmentthereof comprises a VH CDR2 comprising, consisting of, or consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID NO: 114, 115, and 194. In certain embodiments, the antibody orantigen-binding fragment thereof comprises a VH CDR3 comprising,consisting of, or consisting essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO: 15, 34 and 189. In someembodiments, the antibody or antigen-binding fragment thereof comprisesa VL CDR1 comprising, consisting of, or consisting essentially of anamino acid sequence selected from the group consisting of SEQ ID NO: 16and 101-104. In certain embodiments, the antibody or antigen-bindingfragment thereof comprises a VL CDR2 comprising, consisting of, orconsisting essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 17 and 105. In some embodiments, the antibodyor antigen-binding fragment thereof comprises a VL CDR3 comprising,consisting of, or consisting essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO: 18, 106-109, 192, and193. In specific embodiments, the antibody or antigen-binding fragmentthereof comprises the amino acid sequence of the VH CDR1, VH CDR2, andVH CDR3 of an antibody in Table 2. In specific embodiments, the antibodyor antigen-binding fragment thereof comprises the amino acid sequencesof the VH CDR1, VH CDR2, and VH CDR3 of an antibody in Table 6. Inanother specific embodiment, the antibody or antigen-binding fragmentthereof comprises the amino acid sequence of the VL CDR1, VL CDR2, andVL CDR3 of an antibody in Table 1. In another specific embodiment, theantibody or antigen-binding fragment thereof comprises the amino acidsequences of the VL CDR1, VL CDR2, and VL CDR3 of an antibody in Table5. In some embodiments, the antibody or antigen-binding fragment thereofpartially inhibits GITRL (e.g., human GITRL) from binding to GITR (e.g.,human GITR) as assessed by a method known to one of skill in the art ordescribed herein (see, e.g., Sections 6.2.5.2 and 6.2.5.4, infra). Incertain embodiments, the antibody or antigen-binding fragment thereof ata concentration of 1000 ng/ml inhibits less than 80% of 0.5 nM GITRL(e.g., human GITRL) from binding to GITR coupled to beads (e.g., humanGITR coupled to Luminex® beads) at a concentration of 5 pg/ml/beadrelative to the binding of 0.5 nM GITRL to the GITR coupled beads at aconcentration of 5 pg/ml/bead in the absence of the anti-GITR antibodyor antigen-binding fragment thereof in a suspension array assay. Incertain embodiments, the antibody or antigen-binding fragment thereofinhibits 40% to 70%, 50% to 70%, 50% to 80%, or 40% to 80% of the GITRL(e.g., human GITRL) from binding to GITR (e.g., human GITR). In specificembodiments, at least 20% of the amount of GITRL (e.g., human GITRL)that binds to GITR (e.g., human GITR) in the absence of the antibody orantigen-binding fragment thereof binds to GITR (e.g., human GITR) in thepresence of the antibody or antigen-binding fragment thereof in an assaycomprising the following steps: (a) coupling GITR (e.g., human GITR) tobeads at a concentration of 5 pg/ml/bead; (b) incubating the GITR (e.g.,human GITR) coupled beads at a concentration of 40 beads/μl with orwithout the antibody in a well; (c) adding labeled GITRL (e.g., labeledhuman GITRL) to the well to obtain a final concentration of 0.5 nM ofthe GITRL (e.g., human GITRL) 20 beads/μl of the GITR coupled beads; and(d) detecting the labeled GITRL (e.g., human GITRL) bound to the GITR(e.g., human GITR) coupled beads by, e.g., a suspension array assay. Insome embodiments, 20% to 60%, 20% to 50%, 30% to 60% or 30% to 50% ofthe amount of GITRL (e.g., human GITRL) that binds to GITR (e.g., humanGITR) in the absence of the antibody or antigen-binding fragment thereofbinds to GITR (e.g., human GITR) in the presence of the antibody orantigen-binding fragment thereof.

In another embodiment, provided herein is an antibody or antigen-bindingfragment thereof that specifically binds to GITR (e.g., human GITR),comprising:

(a) a heavy chain variable region (VH) CDR1 comprising, consisting of,or consisting essentially of the amino acid sequence of X₁YX₂MX₃ (SEQ IDNO: 7), wherein

X₁ is D, E or G;

X₂ is A or V; and

X₃ is Y or H;

-   -   (b) a VH CDR2 comprising, consisting of, or consisting        essentially of the amino acid sequence of        X₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈(SEQ ID NO: 8), wherein

X₁ is V or L;

X₂ is R, K or Q;

X₃ is Y or F;

X₄ is D, E or G;

X₅ is V or L;

X₆ is T or S;

X₇ is K, R or Q; and

X₈ is D, E or G;

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence of SGTVRGFAY (SEQ ID NO: 9);

(d) a light chain variable region (VL) CDR1 comprising, consisting of,or consisting essentially of the amino acid sequence ofKSSQSLLNSX₁NQKNYLX₂ (SEQ ID NO: 10), wherein

X₁ is G or S; and

X₂ is T or S;

(e) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence of WASTRES (SEQ ID NO: 11); and

(f) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence of QNX₁YSX₂PYT (SEQ ID NO: 12), wherein

X₁ is D or E; and

X₂ is Y, F or S.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises a VH CDR1 comprising, consisting of, or consisting essentiallyof an amino acid sequence selected from the group consisting of SEQ IDNO: 13, 19-23, and 117-119. In certain embodiments, the antibody orantigen-binding fragment thereof comprises a VH CDR1 comprising,consisting of, or consisting essentially of an amino acid sequenceselected from the group consisting of 35 and 116. In some embodiments,the antibody or antigen-binding fragment thereof comprises a VH CDR2comprising, consisting of, or consisting essentially of an amino acidsequence selected from the group consisting of SEQ ID NO: 14, 24-33, and120-188. In some embodiments, the antibody or antigen-binding fragmentthereof comprises a VH CDR2 comprising, consisting of, or consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID NO: 114, 115, and 194. In certain embodiments, the antibody orantigen-binding fragment thereof comprises a VH CDR3 comprising,consisting of, or consisting essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO: 15, 34 and 189. In someembodiments, the antibody or antigen-binding fragment thereof comprisesa VL CDR1 comprising, consisting of, or consisting essentially of anamino acid sequence selected from the group consisting of SEQ ID NO: 16,and 101-104. In certain embodiments, the antibody or antigen-bindingfragment thereof comprises a VL CDR2 comprising, consisting of, orconsisting essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 17 and 105. In some embodiments, the antibodyor antigen-binding fragment thereof comprises a VL CDR3 comprising,consisting of, or consisting essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO: 18, 106-109, 192, and193. In specific embodiments, the antibody or antigen-binding fragmentthereof comprises the amino acid sequence of the VH CDR1, VH CDR2, andVH CDR3 of an antibody in Table 2. In another specific embodiment, theantibody or antigen-binding fragment thereof comprises the amino acidsequence of the VL CDR1, VL CDR2, and VL CDR3 of an antibody in Table 1.In some embodiments, the antibody or antigen-binding fragment thereofdoes not prevent GITRL (e.g., human GITRL) from binding to GITR (e.g.,human GITR) as assessed by a method known to one of skill in the art ordescribed herein (see, e.g., Sections 6.2.5.2 and 6.2.5.4, infra). Incertain embodiments, the antibody or antigen-binding fragment thereof ata concentration of 1000 ng/ml inhibits less than 80% of 0.5 nM GITRL(e.g., human GITRL) from binding to GITR coupled to beads (e.g., humanGITR coupled to Luminex® beads) at a concentration of 5 pg/ml/beadrelative to the binding of 0.5 nM GITRL to the GITR coupled beads at aconcentration of 5 pg/ml/bead in the absence of the anti-GITR antibodyor antigen-binding fragment thereof in a suspension array assay. Incertain embodiments, the antibody or antigen-binding fragment thereofinhibits 40% to 70%, 50% to 70%, 50% to 80%, or 40% to 80% of the GITRL(e.g., human GITRL) from binding to GITR (e.g., human GITR). In specificembodiments, at least 20% of the amount of GITRL (e.g., human GITRL)that binds to GITR (e.g., human GITR) in the absence of the antibody orantigen-binding fragment thereof binds to GITR (e.g., human GITR) in thepresence of the antibody or antigen-binding fragment thereof in an assaycomprising the following steps: (a) coupling GITR (e.g., human GITR) tobeads at a concentration of 5 pg/ml/bead; (b) incubating the GITR (e.g.,human GITR) coupled beads at a concentration of 40 beads/μl with orwithout the antibody in a well; (c) adding labeled GITRL (e.g., labeledhuman GITRL) to the well to obtain a final concentration of 0.5 nM ofthe GITRL (e.g., human GITRL) and 20 beads/μl of the GITR coupled beads;and (d) detecting the labeled GITRL (e.g., human GITRL) bound to theGITR (e.g., human GITR) coupled beads by, e.g., a suspension arrayassay. In some embodiments, 20% to 60%, 20% to 50%, 30% to 60% or 30% to50% of the amount of GITRL (e.g., human GITRL) that binds to GITR (e.g.,human GITR) in the absence of the antibody or antigen-binding fragmentthereof binds to GITR (e.g., human GITR) in the presence of the antibodyor antigen-binding fragment thereof.

In a specific embodiment, provided herein is an antibody or fragmentthereof that specifically binds to human GITR, comprising: (a) a heavychain variable region (VH) CDR1 comprising the amino acid sequence ofDYAMY (SEQ ID NO: 13); (b) a VH CDR2 comprising the amino acid sequenceof VIRTYSGDVTYNQKFKD (SEQ ID NO: 14); (c) a VH CDR3 comprising the aminoacid sequence of SGTVRGFAY (SEQ ID NO: 15); (d) a light chain variableregion (VL) CDR1 comprising the amino acid sequence of KSSQSLLNSGNQKNYLT(SEQ ID NO: 16); (e) a VL CDR2 comprising the amino acid sequence ofWASTRES (SEQ ID NO: 17); and (f) a VL CDR3 comprising the amino acidsequence of QNDYSYPYT (SEQ ID NO: 18). In another embodiment, providedherein is an antibody or fragment thereof that specifically binds tohuman GITR, comprising the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2,and VH CDR 3 of an antibody 22 in Table 1 and Table 2. In someembodiments, the antibody or antigen-binding fragment thereof partiallyinhibits GITRL (e.g., human GITRL) from binding to GITR (e.g., humanGITR) as assessed by a method known to one of skill in the art ordescribed herein (see, e.g., Sections 6.2.5.2 and 6.2.5.4, infra). Incertain embodiments, the antibody or antigen-binding fragment thereof ata concentration of 1000 ng/ml inhibits less than 80% of GITR coupled tobeads (e.g., human GITR coupled to Luminex® beads) at a concentration of5 pg/ml/bead relative to the binding of 0.5 nM GITRL (e.g., human GITRL)to the GITR coupled beads (e.g., human GITR coupled to Luminex® beads)at a concentration of 5 pg/ml/bead in the absence of the anti-GITRantibody or antigen-binding fragment thereof in a suspension arrayassay. In some embodiments, the antibody or antigen-binding fragmentthereof inhibits 50% to 70% of human GITRL binding to human GITR. Inspecific embodiments, at least 20% of the amount of GITRL (e.g., humanGITRL) that binds to GITR (e.g., human GITR) in the absence of theantibody or antigen-binding fragment thereof binds to GITR (e.g., humanGITR) in the presence of the antibody or antigen-binding fragmentthereof in an assay comprising the following steps: (a) coupling GITR(e.g., human GITR) to beads at a concentration of 5 pg/ml/bead; (b)incubating the GITR (e.g., human GITR) coupled beads at a concentrationof 40 beads/μl with or without the antibody in a well; (c) addinglabeled GITRL (e.g., labeled human GITRL) to the well to obtain a finalconcentration of 0.5 nM of the GITRL (e.g., human GITRL) and 20 beads/μlof the GITR coupled beads; and (d) detecting the labeled GITRL (e.g.,human GITRL) bound to the GITR (e.g., human GITR) coupled beads by,e.g., a suspension array assay. In some embodiments, 20% to 60%, 20% to50%, 30% to 60% or 30% to 50% of the amount of GITRL (e.g., human GITRL)that binds to GITR (e.g., human GITR) in the absence of the antibody orantigen-binding fragment thereof binds to GITR (e.g., human GITR) in thepresence of the antibody or antigen-binding fragment thereof. In someembodiments, 30% to 50% of the amount of human GITRL that binds to humanGITR in the absence of the antibody or antigen-binding fragment thereofbinds to human GITR in the presence of the antibody or antigen-bindingfragment thereof. In certain embodiments, the antibody orantigen-binding fragment thereof is agonistic.

In certain embodiments, an antibody or fragment thereof provided herein,which specifically binds to GITR (e.g., human GITR), comprises a heavychain variable region sequence comprising one, two, three or four of theframework regions of the heavy chain variable region sequence of SEQ IDNO: 203. In some embodiments, an antibody or fragment thereof providedherein, which specifically binds to GITR (e.g., human GITR), comprisesone, two, three or four framework regions of a heavy chain variableregion sequence which is at least 75%, 80%, 85%, 90%, 95% or 100%identical to one, two, three or four of the framework regions of a heavychain variable region sequence selected from the group consisting of SEQID NO: 201, SEQ ID NO: 206, and SEQ ID NOs: 215 to 389. In anotherembodiment, an antibody or fragment thereof provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises a heavy chainvariable region having human derived framework regions. In anotherembodiment, an antibody or antigen-binding fragment thereof providedherein, which specifically binds to GITR (e.g., human GITR), comprises aheavy chain variable framework region that is or is derived from anamino acid sequence encoded by a human gene, wherein the amino acidsequence is selected from the group consisting of IGHV1-2*02 (SEQ ID NO:601), IGHV1-3*01 (SEQ ID NO: 602), IGHV1-46*01 (SEQ ID NO: 603),IGHV1-18*01 (SEQ ID NO: 604), IGHV1-69*01 (SEQ ID NO: 605), andIGHV7-4-1*02 (SEQ ID NO: 606). In specific embodiments, the heavy chainvariable framework region that is derived from said amino acid sequenceconsists of said amino acid sequence with up to 10 amino acidsubstitutions, deletions, and/or insertions, preferably up to 10 aminoacid substitutions. In a particular embodiment, the heavy chain variableframework region that is derived from said amino acid sequence consistsof said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aminoacid residues being substituted for an amino acid found in an analogousposition in a corresponding non-human heavy chain variable frameworkregion. In a specific embodiment, an antibody or fragment thereofprovided herein, which specifically binds to GITR (e.g., human GITR),comprises a heavy chain variable framework region that is derived fromamino acid sequence SEQ ID NO: 601, wherein at least one, two, three,four, or five (in certain embodiments up to 10) amino acids of aminoacid sequence SEQ ID NO: 601 is substituted with an amino acid found inan analogous position in a corresponding non-human heavy chain variableframework region. In certain embodiments, the amino acid substitution isat an amino acid position selected from the group consisting of 24, 48,67, 71, 73, and 94, wherein the amino acid position of each group memberis indicated according to the Kabat numbering. In specific embodiments,the amino acid substitution is selected from the group consisting of24G, 48I, 67A, 71V, 73K, and 94K, wherein the amino acid position ofeach group member is indicated according to the Kabat numbering

In another embodiment, provided herein is an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprising aheavy chain variable region sequence comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 201, 206, and SEQ IDNOS: 215 to 389. In a specific embodiment, provided herein is anantibody or fragment thereof that specifically binds to GITR (e.g.,human GITR) comprising a heavy chain variable region sequence comprisingthe amino acid sequence of SEQ ID NO: 203. In another specificembodiment, provided herein is an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprising a heavy chainvariable region sequence comprising the amino acid sequence of SEQ IDNO: 206.

In another embodiment, provided herein is an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprising aheavy chain sequence comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 553, 554 and 567 to 570. In anotherembodiment, provided herein is an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprising a heavy chainsequence comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 581 and 582.

In another embodiment, an antibody or fragment thereof provided herein,which specifically binds to GITR (e.g., human GITR), comprises a lightchain variable region sequence comprising one, two, three or fourframework regions of the light chain variable region sequence of SEQ IDNO: 204 or SEQ ID NO: 205. In another embodiment, an antibody orfragment thereof provided herein, which specifically binds to GITR(e.g., human GITR), comprises one, two, three or four framework regionsof a light chain variable region sequence which is at least 75%, 80%,85%, 90%, 95%, or 100% identical to one, two, three or four of theframework regions of a light chain variable region sequence selectedfrom the group consisting of SEQ ID NO: 202, SEQ ID NO: 207, SEQ ID NO:208, and SEQ ID NOs: 400-518. In another embodiment, an antibody orfragment thereof provided herein, which specifically binds to GITR(e.g., human GITR), comprises one, two, three or four framework regionsof a light chain variable region sequence which is at least 75%, 80%,85%, 90%, 95%, or 100% identical to one, two, three or four of theframework regions of the light chain variable region sequence of SEQ IDNO: 519. In another embodiment, an antibody or fragment thereof providedherein, which specifically binds to GITR (e.g., human GITR), comprises alight chain variable sequence having human derived framework regions. Inanother embodiment, an antibody or fragment thereof provided herein,which specifically binds to GITR (e.g., human GITR), comprises a lightchain variable framework region that is or is derived from an amino acidsequence encoded by a human gene, wherein the amino acid sequence isselected from the group consisting of IGKV4-1*01 (SEQ ID NO: 607) andIGKV3-7*02 (SEQ ID NO: 608). In specific embodiments, the light chainvariable framework region that is derived from said amino acid sequenceconsists of said amino acid sequence but for the presence of up to 10amino acid substitutions, deletions, and/or insertions, preferably up to10 amino acid substitutions. In a particular embodiment, the light chainvariable framework region that is derived from said amino acid sequenceconsists of said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 amino acid residues being substituted for an amino acid found in ananalogous position in a corresponding non-human light chain variableframework region. In another embodiment, an antibody or fragment thereofprovided herein, which specifically binds to GITR (e.g., human GITR),comprises a light chain variable framework region that is or is derivedfrom amino acid sequence SEQ ID NO: 607 or SEQ ID NO: 608 wherein atleast one, two, three, four, or five (in certain embodiments up to 10)amino acids of amino acid sequence SEQ ID NO: 607 or SEQ ID NO: 608 issubstituted with an amino acid found in an analogous position in acorresponding non-human light chain variable framework region. Incertain embodiments, the amino acid substitution is at amino acidposition 87, wherein the amino acid position is indicated according tothe Kabat numbering. In specific embodiments, the amino acidsubstitution is an amino acid substitution of 87H, wherein the aminoacid position is indicated according to the Kabat numbering.

In another embodiment, provided herein is an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprising alight chain variable region sequence comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 202, SEQ ID NO: 207,SEQ ID NO: 208 and SEQ ID NOs: 400 to 518. In another embodiment,provided herein is an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprising a light chain variableregion sequence comprising the amino acid sequence of SEQ ID NO: 519. Ina specific embodiment, provided herein is an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprising alight chain variable region sequence comprising the amino acid sequenceof SEQ ID NO: 204 or SEQ ID NO: 205. In another specific embodiment,provided herein is an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprising a light chain variableregion sequence comprising the amino acid sequence of SEQ ID NO: 207. Inanother specific embodiment, provided herein is an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprising alight chain variable region sequence comprising the amino acid sequenceof SEQ ID NO: 208.

In another embodiment, provided herein is an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprising alight chain sequence comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 555, 556 and 571 to 576.

In another embodiment, provided herein is an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprising aheavy chain variable region and a light chain variable region comprisingthe amino acid sequences of an antibody in the table of FIG. 23 or anyone of FIGS. 24A-24C. In another embodiment, provided herein is anantibody or fragment thereof that specifically binds to GITR (e.g.,human GITR) comprising a heavy chain variable region and a light chainvariable region comprising the amino acid sequences of an antibody inTable 17. In an specific embodiment, provided herein is an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprising (a) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 206; and (b) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 207. In anotherspecific embodiment, provided herein is an antibody or fragment thereofthat specifically binds to GITR (e.g., human GITR) comprising (a) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 206; and (b) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 208.

In a specific embodiment, provided herein is an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprising aheavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, a VHCDR3 and framework regions derived from a human immunoglobulin, whereinVH CDR1 comprises, consists of, or consists essentially of the aminoacid sequence of DYAMY (SEQ ID NO: 13), VH CDR2 comprises, consists of,or consists essentially of the amino acid sequence of VIRTYSGDVTYNQKFKD(SEQ ID NO: 14) and VH CDR3 comprises, consists of, or consistsessentially of the amino acid sequence of SGTVRGFAY (SEQ ID NO: 15). Inanother embodiment, provided herein is an antibody or fragment thereofthat specifically binds to GITR (e.g., human GITR) comprising a lightchain variable region (VL) comprising a VL CDR1, a VL CDR2, a VL CDR3and framework regions derived from a human immunoglobulin, wherein VLCDR1 comprises, consists of, or consists essentially of the amino acidsequence of KSSQSLLNSGNQKNYLT (SEQ ID NO: 16), VL CDR2 comprises,consists of, or consists essentially of the amino acid sequence ofWASTRES (SEQ ID NO: 17) and VL CDR3 comprises, consists of, or consistsessentially of the amino acid sequence of QNDYSYPYT (SEQ ID NO: 18). Inanother embodiment, provided herein is an antibody or fragment thereofthat specifically binds to GITR (e.g., human GITR) comprising a heavychain variable region (VH) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 201, 203, 206 and 215-389. Inanother embodiment, provided herein is an antibody or fragment thereofthat specifically binds to GITR (e.g., human GITR) comprising a lightchain variable region (VL) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 202, 204, 205, 207, 208 and400-518. In another embodiment, provided herein is an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprising a light chain variable region (VL) comprising the amino acidsequence of SEQ ID NO: 519. In some embodiments, the antibody orantigen-binding fragment thereof partially inhibits GITRL (e.g., humanGITRL) from binding to GITR (e.g., human GITR) as assessed by a methodknown to one of skill in the art or described herein (see, e.g.,Sections 6.2.5.2 and 6.2.5.4, infra). In certain embodiments, theantibody or antigen-binding fragment thereof at a concentration of 1000ng/ml inhibits less than 80% of 0.5 nM GITRL (e.g., human GITRL) frombinding to GITR coupled to beads (e.g., human GITR coupled to Luminex®beads) at a concentration of 5 pg/ml/bead relative to the binding of 0.5nM GITRL to the GITR coupled beads at a concentration of 5 pg/ml/bead inthe absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay. In specific embodiments, at least20% of the amount of GITRL (e.g., human GITRL) that binds to GITR (e.g.,human GITR) in the absence of the antibody or antigen-binding fragmentthereof binds to GITR (e.g., human GITR) in the presence of the antibodyor antigen-binding fragment thereof in an assay comprising the followingsteps: (a) coupling GITR (e.g., human GITR) to beads at a concentrationof 5 pg/ml/bead; (b) incubating the GITR (e.g., human GITR) coupledbeads at a concentration of 40 beads/μl with or without the antibody ina well; (c) adding labeled GITRL (e.g., labeled human GITRL) to the wellto obtain a final concentration of 0.5 nM of the GITRL (e.g., humanGITRL) and 20 beads/μl of the GITR coupled beads; and (d) detecting thelabeled GITRL (e.g., human GITRL) bound to the GITR (e.g., human GITR)coupled beads by, e.g., a suspension array assay. In some embodiments,20% to 60%, 20% to 50%, 30% to 60% or 30% to 50% of the amount of GITRL(e.g., human GITRL) that binds to GITR (e.g., human GITR) in the absenceof the antibody or antigen-binding fragment thereof binds to GITR (e.g.,human GITR) in the presence of the antibody or antigen-binding fragmentthereof.

In a specific embodiment, provided herein is an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprising aheavy chain variable region (VH) and a light chain variable region (VL),wherein the VH and VL comprise the amino acid sequence of an antibody inFIG. 23 or any one of FIGS. 24A-24C. In a specific embodiment, providedherein is an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprising a heavy chain variable region (VH)and a light chain variable region (VL), wherein the VH and VL comprisethe amino acid sequence of an antibody in Table 17.

In certain embodiments, an antibody provided herein, which specificallybinds to GITR (e.g., human GITR), comprises heavy and/or light chainconstant regions. In some embodiments, the heavy chain constant regionis selected from the group of human immunoglobulins consisting of IgG₁,IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. In certain embodiments, the lightchain constant region is selected from the group of humanimmunoglobulins consisting of IgGκ and IgGλ. In a specific embodiment,the IgG₁ is non-fucosylated IgG₁. In another specific embodiment, theantibody is an IgG₁ which comprises a N297A or N297Q mutation. Inanother specific embodiment, the antibody is an IgG₄ which comprises aS228P mutation. In another specific embodiment, the antibody is an IgG2which comprises a C127S mutation. In certain embodiments, an antibodyprovided herein, which specifically binds to GITR (e.g., human GITR),comprises a heavy chain constant region comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 557-562. Incertain embodiments, an antibody provided herein, which specificallybinds to GITR (e.g., human GITR), comprises a heavy chain constantregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 583 and 584. In another specific embodiment, anantibody provided herein, which specifically binds to GITR (e.g., humanGITR), comprises a heavy chain constant region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 557-560 withan amino acid substitution of N to A or Q at amino acid position 180. Incertain embodiments, an antibody provided herein, which specificallybinds to GITR (e.g., human GITR), comprises a light chain constantregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 588-591. In certain embodiments, an antibodyprovided herein, which specifically binds to GITR (e.g., human GITR),comprises a light chain constant region comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 563-566.

In a specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 553, 554, and 567 to 570; and (b) a light chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 555, 556, and 571 to 576. In another specific embodiment, anantibody provided herein, which specifically binds to GITR (e.g., humanGITR), comprises (a) a heavy chain comprising the amino acid sequence ofSEQ ID NO: 553; and (b) a light chain comprising the amino acid sequenceof SEQ ID NO: 556. In another specific embodiment, an antibody providedherein, which specifically binds to GITR (e.g., human GITR), comprises(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 554;and (b) a light chain comprising the amino acid sequence of SEQ ID NO:556. In another specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 581; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 556. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 582; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 556. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 553; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 555. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 554; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 555. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 567; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 573. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 567; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 576. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 554; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 576. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 581; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 576. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 582; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 576.

In another specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 553 with an aminoacid substitution of N to A or Q at amino acid position 298; and (b) alight chain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 555, 556, and 571 to 576. In another specificembodiment, an antibody provided herein, which specifically binds toGITR (e.g., human GITR), comprises (a) a heavy chain comprising theamino acid sequence of SEQ ID NO: 553 with an amino acid substitution ofN to A or Q at amino acid position 298; and (b) a light chain comprisingthe amino acid sequence of SEQ ID NO: 556. In another specificembodiment, an antibody provided herein, which specifically binds toGITR (e.g., human GITR), comprises (a) a heavy chain comprising theamino acid sequence of SEQ ID NO: 553 with an amino acid substitution ofN to A or Q at amino acid position 298; and (b) a light chain comprisingthe amino acid sequence of SEQ ID NO: 555.

In another embodiment, provided herein is an antibody or fragmentthereof that binds to the same epitope of GITR (e.g., human GITR) as theantibody described herein. In another embodiment, provided herein is anisolated antibody that specifically binds to each of i) human GITR,wherein the human GITR comprises residues 26-241 of SEQ ID NO: 701 andii) a variant of cynomolgus GITR comprising residues 26-234 of SEQ IDNO: 699, wherein the antibody does not specifically bind to cynomolgusGITR comprising residues 26-234 of SEQ ID NO: 704. In anotherembodiment, provided herein is an isolated antibody that specificallybinds to each of i) human GITR, wherein the human GITR comprisesresidues 26-241 of SEQ ID NO: 701 and ii) a variant of cynomolgus GITRcomprising residues 26-234 of SEQ ID NO: 699, wherein the antibody doesnot exhibit substantial binding to cynomolgus GITR comprising residues26-234 of SEQ ID NO: 704. In another embodiment, provided herein is anisolated antibody that specifically binds to human GITR, wherein thehuman GITR comprises residues 26-241 of SEQ ID NO: 701, wherein thebinding between the antibody and a variant GITR is substantiallyweakened relative to the binding between the antibody and the humanGITR, and wherein the variant GITR comprises residues 26-241 of SEQ IDNO: 701 except for an amino acid substitution selected from the groupconsisting of D60A and G63A. In one embodiment, the substitution isD60A. In another embodiment, the substitution is G63A. In anotherembodiment, provided herein is an isolated antibody that specificallybinds to human GITR, wherein the human GITR comprises residues 26-241 ofSEQ ID NO: 701, and wherein the antibody binds to an epitope comprisingresidues 60-63 of SEQ ID NO: 701. In another embodiment, provided hereinis an isolated antibody that specifically binds to human GITR, whereinthe human GITR comprises residues 26-241 of SEQ ID NO: 701, and whereinthe antibody binds to at least one residue within the amino acidsequence set forth by residues 60-63 of SEQ ID NO: 701. In oneembodiment, the antibody binds to at least one residue selected from thegroup consisting of residues 60, 62, and 63 of SEQ ID NO: 701. In oneembodiment, the antibody binds to at least one residue selected from thegroup consisting of residues 62 and 63 of SEQ ID NO: 701. In oneembodiment, the antibody activates or enhances an activity of the humanGITR. In another embodiment, provided herein is an isolated antibodythat specifically binds human GITR, wherein the human GITR comprisesresidues 26-241 of SEQ ID NO:701, and wherein the antibody binds anepitope of the human GITR comprising at least one of residues 60 or 63of SEQ ID NO:701. In another embodiment provided herein is an isolatedantibody that specifically binds to human GITR, wherein the antibodyexhibits, as compared to binding to human GITR, reduced or absentbinding to a protein identical to human GITR except for the presence ofa D60A or G63A amino acid substitution. In one embodiment, the antibodyinduces, activates or enhances an activity of the human GITR. In anotherembodiment, provided herein is an antibody or antigen-binding fragmentthereof that competes with an antibody or antigen-binding fragmentthereof described herein for binding to GITR (e.g., human GITR). In aspecific embodiment, provided herein is an antibody or antigen-bindingfragment thereof that competes with antibody or antigen-binding fragmentthereof described herein for binding to GITR (e.g., human GITR) to theextent that the antibody or antigen-binding fragment thereof describedherein self-competes for binding to GITR (e.g., human GITR). In anotherspecific embodiment, provided herein is a first antibody orantigen-binding fragment thereof that competes with an antibody orantigen-binding fragment thereof described herein for binding to GITR(e.g., human GITR), wherein the first antibody or antigen-bindingfragment thereof competes for binding in an assay comprising thefollowing steps: (a) incubating GITR-transfected cells with the firstantibody or antigen-binding fragment thereof in unlabeled form in acontainer; (b) adding the antibody or antigen-binding fragment thereofdescribed herein in labeled form to the cells in the container andincubating the cells in the container; and (c) detecting the binding ofthe antibody or antigen-binding fragment thereof described herein inlabeled form to the cells. In another specific embodiment, providedherein is a first antibody or antigen-binding fragment thereof competeswith an antibody or antigen-binding fragment thereof described hereinfor binding to GITR (e.g., human GITR), wherein the competition isexhibited as reduced binding of first antibody or antigen-bindingfragment thereof to GITR (e.g., human GITR) by more than 80% (e.g., 85%,90%, 95%, or 98%, or between 80% to 85%, 80% to 90%, 85% to 90%, or 85%to 95%).

In certain embodiments, an antibody or fragment thereof provided herein,which specifically binds to GITR (e.g., human GITR), activates, inducesor enhances an activity of GITR (e.g., human GITR). In specificembodiments, an antibody provided herein, which specifically binds toGITR (e.g., human GITR), is a humanized antibody, murine antibody orchimeric antibody. In certain embodiments, an antibody provided herein,which specifically binds to GITR (e.g., human GITR), binds to GITR(e.g., human GITR) with a K_(D) in the range of about 0.5 nM to 5 nM. Incertain embodiments, an antibody provided herein, which specificallybinds to GITR (e.g., human GITR), comprises a detectable label. Inspecific embodiments, an antibody provided herein is isolated.

In certain embodiments, an antibody or fragment described herein, whichimmunospecifically binds to GITR (e.g., human GITR), induces, activatesor enhances an activity of human GITR in a cell independent of TCRtriggering. In specific embodiments, the cell is a T cell. In specificembodiments, the cell is not a T cell. In specific embodiments, the cellis selected from the group consisting of a B cell, a plasma cell, amemory cell, a natural killer cell, a granulocyte, a neutrophil, aneosinophil, a basophil, a mast cell, a monocyte, a dendritic cell, aplasmacytoid dendritic cell, an NKT cell, and a macrophage. In specificembodiments, an antibody described herein, which immunospecificallybinds to GITR (e.g., human GITR), induces, activates, or enhances anactivity of NF-κB independent of TCR triggering. In certain embodiments,the activity of NF-κB can be assessed in, e.g., an assay comprising thefollowing steps: (a) incubating T cells (e.g., Jurkat cells) expressinga NF-κB-luciferase reporter construct (e.g., GloResponse NF-κB-luc2Pconstruct) and GITR (e.g., human GITR) with the antibody describedherein or an isotype control antibody at an antibody concentration of,e.g., 12.5, 10, 5, 2.5, 1.25, or 0.625 μg/ml, in the absence of ananti-CD3 antibody; and (b) reading luciferase signal after, e.g., 2, 5,6, 8 or 18 hours of incubation using, e.g., an EnVision multilabelreader 2100, wherein a positive luciferase signal relative to theisotype control antibody indicates activity of NF-κB. In a particularembodiment, the luciferase signal is read after 5 hours of incubation.

In certain embodiments, an antibody or fragment described herein, whichimmunospecifically binds to GITR (e.g., human GITR), increases thepercentage of polyfunctional (IFNγ+ TNFα+) T cells. In specificembodiments, the increase in the percentage of polyfunctional (IFNγ+TNFα+) T cells can be assessed in, e.g., an assay comprising thefollowing steps: (a) incubating, e.g., human PBMCs with, e.g., ananti-CD3 antibody at various suboptimal concentrations (e.g., 0.3-5μg/ml); and, e.g., an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), at, e.g., 5 μg/mlor an isotype control antibody, for, e.g., 3-4 days at 37° C. and 5%CO₂; (b) treating cells with, e.g., Brefeldin A for, e.g., 6 hours at37° C. and 5% CO₂; (c) staining the surface of the cells using, e.g., ananti-CD3 antibody, an anti-CD4 antibody, and an anti-CD8α antibody; (d)staining intracellularly using, e.g., an anti-IFNγ antibody and ananti-TNFα antibody; and (e) determining the percentage of polyfunctional(IFNγ+ TNFα+) T cells relative to the isotype control antibody. Inspecific embodiments, the polyfunctional (IFNγ+ TNFα+) T cells areselected from the group consisting of polyfunctional (IFNγ+ TNFα+) CD4+T cells and polyfunctional (IFNγ+ TNFα+) CD8+ T cells.

In specific embodiments, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), when bound toactivated regulatory T cells, binds to activating Fc gamma receptorsselected from the group consisting of CD16, CD32A and CD64 to a greaterextent (e.g., 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold,3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, or 100 fold) than the antibody, when bound toactivated effector T cells, binds to the activating Fc gamma receptorsselected from the group consisting of CD16, CD32A and CD64, as assessedby methods described herein or known to one of skill in the art (e.g.,an Fc gamma receptor IIIA (CD16) reporter assay or as described in theExamples, infra). In specific embodiments, the activating Fc gammareceptors are expressed on a cell selected from the group consisting ofmyeloid-derived effector cells and lymphocyte-derived effector cells. Ina particular embodiment, the activating Fc gamma receptor is CD16.

In specific embodiments, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), when bound toactivated regulatory T cells, causes stronger activation of activatingFc gamma receptors selected from the group consisting of CD16, CD32A andCD64 than the antibody, when bound to activated effector T cells, causesactivation of activating Fc gamma receptors selected from the groupconsisting of CD16, CD32A and CD64. In particular embodiments, theactivation of the activating Fc gamma receptors, when the antibodydescribed herein, which immunospecifically binds to GITR (e.g., humanGITR), is bound to activated regulatory T cells, is at least about 1.2fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold,4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold stronger than the activation of the activating Fcgamma receptors, when the antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), is bound toactivated effector T cells, as assessed by methods described herein orknown to one of skill in the art (e.g., an Fc gamma receptor IIIA (CD16)reporter assay or as described in the Examples, infra). In specificembodiments, the activating Fc gamma receptors are expressed on a cellselected from the group consisting of myeloid-derived effector cells andlymphocyte-derived effector cells. In a particular embodiment, theactivating Fc gamma receptor is CD16.

In certain embodiments, an antibody or fragment described herein, whichimmunospecifically binds to GITR (e.g., human GITR), increases surfaceexpression of OX40 and/or PD-1 in activated T cells by at least about1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold,15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold,90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700fold, 800 fold, 900 fold, or 1000 fold as assessed by methods describedherein and/or known to one of skill in the art, relative to surfaceexpression of OX40 and/or PD-1 in activated T cells without the antibodydescribed herein.

In another embodiment, provided herein are nucleic acid moleculesencoding a heavy chain variable region and/or a light chain variableregion, or a light chain and/or a heavy chain of an antibody describedherein. In a specific embodiment, the nucleic acid molecule encodes aheavy chain variable region comprising the nucleic acid sequence of SEQID NO: 209. In another specific embodiment, the nucleic acid moleculeencodes a light chain variable region comprising the nucleic acidsequence of SEQ ID NO: 210 or SEQ ID NO: 211. In specific embodiments,the nucleic acid molecule is isolated. In certain embodiments, a vector(e.g., an isolated vector) comprises a nucleic acid molecule encoding aheavy chain variable region and/or a light chain variable region, or alight chain and/or a heavy chain of an antibody described herein. Incertain embodiments, a host cell comprises the nucleic acid molecule orvector. Examples of host cells include E. coli, Pseudomonas, Bacillus,Streptomyces, yeast, CHO, YB/20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa,BHK, Hep G2, SP2/0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10cells, plant cells, insect cells, and human cells in tissue culture. Ina specific embodiment, provided herein is a method of producing anantibody or antigen-binding fragment thereof that specifically binds toGITR (e.g., human GITR) comprising culturing a host cell so that thenucleic acid molecule is expressed and the antibody is produced.

In another embodiment, provided herein is a method for enhancing theco-stimulation of T cells comprising incubating ex vivo T cells, whichhave been stimulated with a T cell receptor (TCR) complex stimulatingagent (e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate(PMA), or a TCR complex stimulating antibody, such as an anti-CD3antibody and anti-CD28 antibody), with an antibody or antigen-bindingfragment thereof described herein. In another embodiment, providedherein is a method for activating T cells comprising incubating ex vivoT cells with an antibody or antigen-binding fragment thereof describedherein. In some embodiments, the method further comprises, prior to,simultaneously with, or subsequent to the incubation with the anti-GITRantibody or antigen-binding fragment thereof, incubating ex vivo the Tcells with a TCR complex stimulating agent (e.g., phytohaemagglutinin(PHA) and/or phorbol myristate acetate (PMA), or a TCR complexstimulating antibody, such as an anti-CD3 antibody and anti-CD28antibody). In some embodiments, the T cells were isolated from asubject. In certain embodiments, the stimulated and/or activated T cellsare infused into a subject. In some embodiments, the T cells beinginfused into the subject are autologous or allogenic. In a specificembodiment, the subject is a human. In another embodiment, providedherein is a method for preferential expansion of effector T-cells overthat of T-regulatory cells in a subject, comprising administering to thesubject an effective amount of an antibody or antigen-binding fragmentthereof described herein. In a specific embodiment, the subject ishuman.

In another embodiment, provided herein is a method for enhancing theexpansion of T cells (e.g., CD4⁺ and/or CD8⁺ T cells) and/or T celleffector function, comprising incubating ex vivo the T cells with anantibody or antigen-binding fragment thereof described herein. In someembodiments, the method further comprises, prior to, simultaneously withor subsequent to incubating the T cells with the antibody orantigen-binding fragment thereof, incubating the T cells with a T cellreceptor (TCR) complex stimulating agent (e.g., phytohaemagglutinin(PHA) and/or phorbol myristate acetate (PMA), or a TCR complexstimulating antibody, such as an anti-CD3 antibody and anti-CD28antibody). In certain embodiments, the T cells were isolated from asubject. In some embodiments, the method further comprises infusing theT cells after their expansion and/or after their effector function isenhanced into a subject. In certain embodiments, the T cells beinginfused into the subject are autologous or allogenic. In a specificembodiment, the subject is a human.

In another embodiment, provided herein is a method for enhancing theexpansion of CD8⁺ T cells, comprising incubating ex vivo the T cellswith an antibody or antigen-binding fragment thereof described herein.In some embodiments, the method further comprises, prior to,simultaneously with or subsequent to incubating the T cells with theantibody or antigen-binding fragment thereof, incubating the T cellswith a T cell receptor (TCR) complex stimulating agent (e.g.,phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody). In certain embodiments, the T cells were isolatedfrom a subject. In some embodiments, the method further comprisesinfusing the T cells after their expansion and/or after their effectorfunction is enhanced into a subject. In certain embodiments, the T cellsbeing infused into the subject are autologous or allogenic. In aspecific embodiment, the subject is a human.

In another embodiment, provided herein is a method for enhancing theexpansion of CD4⁺ T cells, comprising incubating ex vivo the T cellswith an antibody or antigen-binding fragment thereof described herein.In some embodiments, the method further comprises, prior to,simultaneously with or subsequent to incubating the T cells with theantibody or antigen-binding fragment thereof, incubating the T cellswith a T cell receptor (TCR) complex stimulating agent (e.g.,phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody). In certain embodiments, the T cells were isolatedfrom a subject. In some embodiments, the method further comprisesinfusing the T cells after their expansion and/or after their effectorfunction is enhanced into a subject. In certain embodiments, the T cellsbeing infused into the subject are autologous or allogenic. In aspecific embodiment, the subject is a human.

In another embodiment, provided herein is a method of activating GITR oractivating NF-κB comprising incubating ex vivo T cells, which have notbeen stimulated with a T cell receptor (TCR) complex stimulating agent(e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA),or a TCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody), with an antibody or antigen-binding fragmentthereof described herein. In some embodiments, the T cells were isolatedfrom a subject. In certain embodiments, the activated T cells areinfused into a subject. In some embodiments, the T cells being infusedinto the subject are autologous or allogenic. In a specific embodiment,the subject is a human.

In another embodiment, provided herein is a method of activating T cellsindependent of TCR triggering comprising contacting T cells with anantibody or antigen-binding fragment thereof described herein.

In another embodiment, provided herein is a method of inducing,activating or enhancing an activity of NF-κB independent of TCRtriggering comprising contacting T cells with an antibody orantigen-binding fragment thereof described herein.

In another embodiment, provided herein is a method of increasing thepercentage of polyfunctional (IFNγ+ TNFα+) T cells comprising contactingT cells with an antibody or antigen-binding fragment thereof describedherein.

In another embodiment, provided herein is a method of increasing surfaceexpression of OX40 and/or PD-1 in activated T cells comprisingcontacting T cells with an antibody or antigen-binding fragment thereofdescribed herein.

In another embodiment, provided herein are pharmaceutical compositionscomprising an antibody or antigen-binding fragment thereof, a nucleicacid molecule, a vector, or a host cell described herein, and apharmaceutically acceptable carrier. The pharmaceutical composition canbe used to modulate immune response and/or to treat and/or prevent adisorder, such as cancer or an infectious disease. In a specificembodiment, provided herein is a method of modulating an immune responsein a subject, comprising administering to the subject an effectiveamount of the pharmaceutical composition described herein. In aparticular embodiment, the immune response is enhanced or induced. Inanother specific embodiment, provided herein is a method for enhancingthe expansion of T cells and/or T cell effector function in a subject,comprising administering to the subject an effective amount of apharmaceutical composition described herein. In another specificembodiment, provided herein is a method for enhancing the expansion ofCD8+ T cells in a subject, comprising administering to the subject aneffective amount of a pharmaceutical composition described herein. Insome embodiments, the disclosure provides use of an antibody asdescribed herein in the manufacture of a medicament for the treatment ofcancer. In certain embodiments, the disclosure provides an antibody asdescribed herein for use in the treatment of cancer. In certainembodiments, the disclosure provides use of a pharmaceutical compositionas described herein in the manufacture of a medicament for the treatmentof cancer. In certain embodiments, the disclosure provides apharmaceutical composition as described herein for use in the treatmentof cancer. In another specific embodiment, provided herein is a methodof treating cancer in a subject, comprising administering to the subjectan effective amount of the pharmaceutical composition described herein.In certain embodiments, the method of treating cancer further comprisesadministering an anti-cancer agent to the subject. Examples ofanti-cancer agents that can be administered to a subject in combinationwith a pharmaceutical composition described herein are described inSection 5.4, infra (e.g., Sections 5.4.1 and 5.4.1.1). In a specificembodiment, the anti-cancer agent is a vaccine. In a particularembodiment, the vaccine comprises a heat shock protein peptide complex(HSPPC), in which the HSPPC comprises a heat shock protein (e.g., a gp96protein) complexed with one or more antigenic peptides (e.g.,tumor-associated antigenic peptides). In certain embodiments, the cancertreated is squamous cell cancer, small-cell lung cancer, non-small celllung cancer, gastrointestinal cancer, Hodgkin's or non-Hodgkin'slymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, myeloma, salivarygland carcinoma, kidney cancer, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, or a type of head or neck cancer. In certain embodiments, thecancer treated is desmoplastic melanoma, inflammatory breast cancer,thymoma, rectal cancer, anal cancer, or surgically treatable ornon-surgically treatable brain stem glioma. In a specific embodiment,the subject treated is a human.

In another embodiment, provided herein is a method for activating Tcells independent of TCR triggering in a subject, comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition described herein.

In another embodiment, provided herein is a method for inducing,activating or enhancing an activity of NF-κB independent of TCRtriggering in a subject, comprising administering to the subject aneffective amount of the pharmaceutical composition described herein.

In another embodiment, provided herein is a method for increasing thepercentage of polyfunctional (IFNγ+ TNFα+) T cells in a subject,comprising administering to the subject an effective amount of thepharmaceutical composition described herein.

In another embodiment, provided herein is a method for increasingsurface expression of OX40 and/or PD-1 in activated T cells in asubject, comprising administering to the subject an effective amount ofthe pharmaceutical composition described herein.

The antibody as described herein can be used in combination with an IDOinhibitor for treating cancer. In one embodiment, provided herein is amethod of treating cancer in a subject, comprising administering to thesubject an effective amount of the pharmaceutical composition describedherein, wherein the method further comprises administering to thesubject an inhibitor of indoleamine-2,3-dioxygenase (IDO). The IDOinhibitor as described herein for use in treating cancer is present in asolid dosage form of a pharmaceutical composition such as a tablet, apill or a capsule, wherein the pharmaceutical composition includes anIDO inhibitor and a pharmaceutically acceptable excipient. As such, theantibody as described herein and the IDO inhibitor as described hereincan be administered separately, sequentially or concurrently as separatedosage forms. In one embodiment, the antibody is administeredparenterally and the IDO inhibitor is administered orally. In particularembodiments, the inhibitor is selected from the group consisting ofepacadostat (Incyte Corporation), F001287 (Flexus Biosciences),indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). Epacadostathas been described in PCT Publication No. WO 2010/005958, which isincorporated herein by reference in its entirety for all purposes. Inone embodiment, the inhibitor is epacadostat. In another embodiment, theinhibitor is F001287. In another embodiment, the inhibitor is indoximod.In another embodiment, the inhibitor is NLG919.

The antibody as described herein can be used in combination with acheckpoint targeting agent for treating cancer. In one embodiment,provided herein is a method of treating cancer in a subject, comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition described herein, wherein the method further comprisesadministering to the subject a checkpoint targeting agent. In someembodiments, the checkpoint targeting agent is selected from the groupconsisting of an antagonist of PD-1 (e.g., an antagonist anti-PD-1antibody), an antagonist of PD-L1 (e.g., an antagonist anti-PD-L1antibody), an antagonist of PD-L2 (e.g., an antagonist anti-PD-L2antibody), an antagonist of CTLA-4 (e.g., an antagonist anti-CTLA-4antibody), an antagonist of TIM-3 (e.g., an antagonist anti-TIM-3antibody), an antagonist of LAG-3 (e.g., an antagonist anti-LAG-3antibody), and an agonist of OX40 (e.g., an agonist anti-OX40 antibody).In some embodiments, a checkpoint targeting agent, e.g., an antagonistof PD-1 (e.g., an antagonist anti-PD-1 antibody) or an agonist of OX40(e.g., an agonist anti-OX40 antibody) is administered simultaneouslywith the anti-GITR antibody. In some embodiments, a checkpoint targetingagent, e.g., an antagonist of PD-1 (e.g., an antagonist anti-PD-1antibody) or an agonist of OX40 (e.g., an agonist anti-OX40 antibody) isadministered prior to the administration of the anti-GITR antibody. Insome embodiments, a checkpoint targeting agent, e.g., an antagonist ofPD-1 (e.g., an antagonist anti-PD-1 antibody) or an agonist of OX40(e.g., an agonist anti-OX40 antibody) is administered subsequent to theadministration of the anti-GITR antibody.

The antibody as described herein can be used in combination with ananti-CD25 antibody. In some embodiments, an anti-CD25 antibody isadministered simultaneously with the anti-GITR antibody. In someembodiments, an anti-CD25 antibody is administered prior to theadministration of the anti-GITR antibody. In some embodiments, ananti-CD25 antibody is administered subsequent to the administration ofthe anti-GITR antibody.

In another specific embodiment, provided herein is a method of treatingcancer in a subject comprising administering an antagonist anti-PD-1antibody to a subject in need thereof who has received an anti-GITRantibody, wherein the PD-1 antibody is administered at a time at whichthe anti-GITR antibody has increased expression of PD-1 in the subjectrelative to expression of PD-1 in the subject at the time of theadministering. In another specific embodiment, provided herein is amethod of treating cancer in a subject comprising administering anagonist anti-OX40 antibody to a subject in need thereof who has receivedan anti-GITR antibody, wherein the OX40 antibody is administered at atime at which the anti-GITR antibody has increased expression of OX40 inthe subject relative to expression of OX40 in the subject at the time ofthe administering. In certain embodiments, the anti-GITR antibodyinduces, activates, or enhances an activity of GITR.

In another specific embodiment, provided herein is a method of treatingcancer in a subject comprising administering an anti-GITR antibody to asubject in need thereof, wherein the anti-GITR antibody increasesexpression of PD-1 in the subject relative to expression of PD-1 in thesubject at the time of the administering, and administering anantagonist anti-PD-1 antibody to the subject when expression of PD-1 isincreased. In another specific embodiment, provided herein is a methodof treating cancer in a subject comprising administering an anti-GITRantibody to a subject in need thereof, wherein the anti-GITR antibodyincreases expression of OX40 in the subject relative to expression ofOX40 in the subject at the time of the administering, and administeringan agonist OX40 antibody to the subject when expression of OX40 isincreased. In certain embodiments, the anti-GITR antibody induces,activates, or enhances an activity of GITR.

In another specific embodiment, provided herein is a method of treatingcancer or a viral infection in a subject, the method comprising thesteps of: (a) incubating T cells ex vivo with an antibody orantigen-binding fragment thereof described herein; and (b) infusing theT cells into the subject. In a specific embodiment, the T cells infusedinto the subject are autologous or allogenic. In certain embodiments,the T cells were isolated from a subject. In some embodiments, the Tcells are not incubated with a T cell receptor (TCR) complex stimulatingagent (e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate(PMA), or a TCR complex stimulating antibody, such as an anti-CD3antibody and anti-CD28 antibody). In certain embodiments, the methodfurther comprises, prior to step (a): (1) assaying the T cells for cellsurface expression of GITR; and (2) if step (1) does not result indetection of GITR above a threshold value, inducing expression of GITRon the surface of the T cells by incubating the T cells with a T cellreceptor (TCR) complex stimulating agent (e.g., phytohaemagglutinin(PHA) and/or phorbol myristate acetate (PMA), or a TCR complexstimulating antibody, such as an anti-CD3 antibody and anti-CD28antibody). In some embodiments, the method further comprises, prior to,simultaneously with or subsequent to step (a), incubating the T cellswith a T cell receptor (TCR) complex stimulating agentphytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody). In a specific embodiment, the subject treated is ahuman.

In another embodiment, provided herein is a method of treating and/orpreventing an infectious disease in a subject comprising administeringto the subject an effective amount of a pharmaceutical compositiondescribed herein. See Section 5.4.1.2 below for examples of infectiousdiseases. In another specific embodiment, provided herein is a method oftreating a viral infection in a subject, comprising administering to thesubject an effective amount of the pharmaceutical composition describedherein. In certain embodiments, the viral infection treated is caused bya human papilloma virus (HPV), a Herpes simplex or other herpes virus,hepatitis B virus (HBV), hepatitis C virus (HCV) or other hepatitisvirus, measles virus, HIV or Epstein Barr virus (EBV). In certainembodiments, the method of treating a viral infection further comprisesadministering an anti-viral agent to the subject. In a specificembodiment, the subject treated is a human.

In another specific embodiment, provided herein is a method ofidentifying an anti-GITR antibody that is capable of inducing,activating, or enhancing an activity of GITR in the absence of a TCRagonist comprising contacting a cell expressing GITR with an anti-GITRantibody in the absence of a TCR agonist and measuring GITR activity,wherein increased GITR activity compared to GITR activity in the absenceof the anti-GITR antibody indicates the anti-GITR antibody is capable ofinducing, activating, or enhancing an activity of GITR in the absence ofa TCR agonist. In certain embodiments, the GITR activity is assessed bymeasuring NF-κB activity. In certain embodiments, the GITR activity isassessed by measuring activation of TRAF adaptor mediated signalingpathways, wherein the TRAF adaptor is selected from the group consistingof TRAF1, TRAF2, TRAF3, TRAF4, and TRAF5. In certain embodiments, theGITR activity is assessed by measuring activation of the MAPK/ERKpathway. In certain embodiments, the anti-GITR antibody increases theGITR activity at least two-fold compared to GITR activity in the absenceof the anti-GITR antibody. In certain embodiments, the anti-GITRantibody increases the GITR activity two-fold to twenty-fold compared toGITR activity in the absence of the anti-GITR antibody. In certainembodiments, the anti-GITR antibody increases the GITR activity two-foldto ten-fold compared to GITR activity in the absence of the anti-GITRantibody. In certain embodiments, the cell is a T cell. In certainembodiments, the cell is not a T cell.

In another specific embodiment, provided herein is an anti-GITR antibodythat specifically binds to human GITR, wherein said antibody is capableof inducing, activating, or enhancing an activity of GITR in a cell inthe absence of TCR triggering. In another specific embodiment, providedherein is an anti-GITR antibody that specifically binds to human GITR,wherein said antibody induces, activates, or enhances an activity ofNF-κB in a cell in the absence of TCR triggering. In another specificembodiment, provided herein is a method of treating cancer comprisingadministering to a subject in need thereof an anti-GITR antibody thatspecifically binds to human GITR, wherein said antibody is capable ofinducing, activating, or enhancing an activity of GITR and/or NF-κB inthe absence of TCR triggering.

3.1 Terminology

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above and 5% to 10% below the value or range remain within theintended meaning of the recited value or range.

As used herein, the binding between a test antibody and a first antigenis “substantially weakened” relative to the binding between the testantibody and a second antigen if the binding between the test antibodyand the first antigen is reduced by at least 30%, 40%, 50%, 60%, 70%, or80% relative to the binding between the test antibody and the secondantigen, e.g., in a given experiment, or using mean values from multipleexperiments, as assessed by, e.g., an assay comprising the followingsteps: (a) expressing on the surface of cells (e.g., 1624-5 cells) thefirst antigen or the second antigen; (b) staining the cells expressingthe first antigen or the second antigen using, e.g., 2 μg/ml of the testantibody or a polyclonal antibody in a flow cytometry analysis andrecording mean fluorescence intensity (MFI) values, e.g., as the meanfrom more than one measurement, wherein the polyclonal antibodyrecognizes both the first antigen and the second antigen; (c) dividingthe MFI value of the test antibody for the cells expressing the secondantigen by the MFI value of the polyclonal antibody for the cellsexpressing the second antigen (MFI ratio₂); (d) dividing the MFI valueof the test antibody for the cells expressing the first antigen by theMFI value of the polyclonal antibody for the cells expressing the firstantigen (MFI ratio₁); and (e) determining the percentage of reduction inbinding by calculating 100%*(1−(MFI ratio₁/MFI ratio₂)).

As used herein, an antibody does not exhibit “substantial binding” to anantigen if when measured in a flow cytometry analysis, the meanfluorescence intensity (MFI) value of the antibody to the antigen is notsignificantly higher than the MFI value of an isotype control antibodyto the antigen or the MFI value in the absence of any antibody.

As used herein, the terms “antibody” and “antibodies” are terms of artand can be used interchangeably herein and refer to a molecule with anantigen binding site that specifically binds an antigen.

Antibodies can include, for example, monoclonal antibodies,recombinantly produced antibodies, monospecific antibodies,multispecific antibodies (including bispecific antibodies), humanantibodies, humanized antibodies, chimeric antibodies, immunoglobulins,synthetic antibodies, tetrameric antibodies comprising two heavy chainand two light chain molecules, an antibody light chain monomer, anantibody heavy chain monomer, an antibody light chain dimer, an antibodyheavy chain dimer, an antibody light chain-antibody heavy chain pair,intrabodies, heteroconjugate antibodies, single domain antibodies,monovalent antibodies, single chain antibodies or single-chain Fvs(scFv), camelized antibodies, affybodies, Fab fragments, F(ab′)₂fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id)antibodies (including, e.g., anti-anti-Id antibodies), andantigen-binding fragments of any of the above. In certain embodiments,antibodies described herein refer to polyclonal antibody populations.Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY),any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ or IgA₂), or any subclass(e.g., IgG_(2a) or IgG_(2b)) of immunoglobulin molecule. In certainembodiments, antibodies described herein are IgG antibodies, or a class(e.g., human IgG₁ or IgG₄) or subclass thereof. In a specificembodiment, the antibody is a humanized monoclonal antibody. In anotherspecific embodiment, the antibody is a human monoclonal antibody,preferably that is an immunoglobulin. In certain embodiments, anantibody described herein is an IgG₁, or IgG₄ antibody.

As used herein, the terms “antigen-binding domain,” “antigen-bindingregion,” “antigen-binding fragment,” and similar terms refer to aportion of an antibody molecule which comprises the amino acid residuesthat confer on the antibody molecule its specificity for the antigen(e.g., the complementarity determining regions (CDR)). Theantigen-binding region can be derived from any animal species, such asrodents (e.g., mouse, rat or hamster) and humans.

As used herein, the terms “variable region” or “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 120amino acids in the mature heavy chain and about 90 to 115 amino acids inthe mature light chain, which differ extensively in sequence amongantibodies and are used in the binding and specificity of a particularantibody for its particular antigen. The variability in sequence isconcentrated in those regions called complementarity determining regions(CDRs) while the more highly conserved regions in the variable domainare called framework regions (FR). Without wishing to be bound by anyparticular mechanism or theory, it is believed that the CDRs of thelight and heavy chains are primarily responsible for the interaction andspecificity of the antibody with antigen. In certain embodiments, thevariable region is a human variable region. In certain embodiments, thevariable region comprises rodent or murine CDRs and human frameworkregions (FRs). In particular embodiments, the variable region is aprimate (e.g., non-human primate) variable region. In certainembodiments, the variable region comprises rodent or murine CDRs andprimate (e.g., non-human primate) framework regions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

The term “Kabat numbering” and like terms are recognized in the art andrefer to a system of numbering amino acid residues in the heavy andlight chain variable regions of an antibody, or an antigen-bindingportion thereof. In certain aspects, the CDRs of an antibody can bedetermined according to the Kabat numbering system (see, e.g., Kabat E A& Wu T T (1971) Ann NY Acad Sci 190: 382-391 and Kabat E A et al.,(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242). Using the Kabat numbering system, CDRs within an antibodyheavy chain molecule are typically present at amino acid positions 31 to35, which optionally can include one or two additional amino acids,following 35 (referred to in the Kabat numbering scheme as 35A and 35B)(CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions95 to 102 (CDR3). Using the Kabat numbering system, CDRs within anantibody light chain molecule are typically present at amino acidpositions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), andamino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRsof the antibodies described herein have been determined according to theKabat numbering scheme.

As used herein, the term “constant region” or “constant domain” areinterchangeable and have its meaning common in the art. The constantregion is an antibody portion, e.g., a carboxyl terminal portion of alight and/or heavy chain which is not directly involved in binding of anantibody to antigen but which can exhibit various effector functions,such as interaction with the Fc receptor. The constant region of animmunoglobulin molecule generally has a more conserved amino acidsequence relative to an immunoglobulin variable domain.

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃ and IgG₄.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art. In specific embodiments,the light chain is a human light chain.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured and/or expressedin a number of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D)), and equilibrium associationconstant (K_(A)). The K_(D) is calculated from the quotient ofk_(off)/k_(on), whereas K_(A) is calculated from the quotient ofk_(on)/k_(off). k_(on) refers to the association rate constant of, e.g.,an antibody to an antigen, and k_(off) refers to the dissociation of,e.g., an antibody to an antigen. The k_(on) and k_(off) can bedetermined by techniques known to one of ordinary skill in the art, suchas BIAcore® or KinExA.

As used herein, a “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Incertain embodiments, one or more amino acid residues within a CDR(s) orwithin a framework region(s) of an antibody or antigen-binding fragmentthereof can be replaced with an amino acid residue with a similar sidechain.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody can specificallybind. An epitope can be, for example, contiguous amino acids of apolypeptide (linear or contiguous epitope) or an epitope can, forexample, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope to whichan antibody binds can be determined by, e.g., NMR spectroscopy, X-raydiffraction crystallography studies, ELISA assays, hydrogen/deuteriumexchange coupled with mass spectrometry (e.g., liquid chromatographyelectrospray mass spectrometry), array-based oligo-peptide scanningassays, and/or mutagenesis mapping (e.g., site-directed mutagenesismapping). For X-ray crystallography, crystallization may be accomplishedusing any of the known methods in the art (e.g., Giegé R et al., (1994)Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A(1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303).Antibody:antigen crystals may be studied using well known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H W etal.; U.S. 2004/0014194), and BUSTER (Bricogne G (1993) Acta CrystallogrD Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies may beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and CunninghamB C & Wells J A (1989) Science 244: 1081-1085 for a description ofmutagenesis techniques, including alanine scanning mutagenesistechniques. In a specific embodiment, the epitope of an antibody orantigen-binding fragment thereof is determined using alanine scanningmutagenesis studies, such as described in Section 6, infra.

As used herein, the terms “immunospecifically binds,”“immunospecifically recognizes,” “specifically binds,” and “specificallyrecognizes” are analogous terms in the context of antibodies and referto molecules that bind to an antigen (e.g., epitope or immune complex)as such binding is understood by one skilled in the art. For example, amolecule that specifically binds to an antigen may bind to otherpeptides or polypeptides, generally with lower affinity as determinedby, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (SapidyneInstruments, Boise, Id.), or other assays known in the art. In aspecific embodiment, molecules that immunospecifically bind to anantigen bind to the antigen with a K_(A) that is at least 2 logs, 2.5logs, 3 logs, 4 logs or greater than the K_(A) when the molecules bindto another antigen.

In another specific embodiment, molecules that immunospecifically bindto an antigen do not cross react with other proteins under similarbinding conditions. In another specific embodiment, molecules thatimmunospecifically bind to an antigen do not cross react with othernon-GITR proteins. In a specific embodiment, provided herein is anantibody or fragment thereof that binds to GITR with higher affinitythan to another unrelated antigen. In certain embodiments, providedherein is an antibody or fragment thereof that binds to GITR (e.g.,human GITR) with a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or higher affinity than to another,unrelated antigen as measured by, e.g., a radioimmunoassay, surfaceplasmon resonance, or kinetic exclusion assay. In a specific embodiment,the extent of binding of an anti-GITR antibody or antigen-bindingfragment thereof described herein to an unrelated, non-GITR protein isless than 10%, 15%, or 20% of the binding of the antibody to GITRprotein as measured by, e.g., a radioimmunoassay.

In a specific embodiment, provided herein is an antibody or fragmentthereof that binds to human GITR with higher affinity than to anotherspecies of GITR. In certain embodiments, provided herein is an antibodyor fragment thereof that binds to human GITR with a 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher affinity thanto another species of GITR as measured by, e.g., a radioimmunoassay,surface plasmon resonance, or kinetic exclusion assay. In a specificembodiment, an antibody or fragment thereof described herein, whichbinds to human GITR, will bind to another species of GITR protein withless than 10%, 15%, or 20% of the binding of the antibody or fragmentthereof to the human GITR protein as measured by, e.g., aradioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.

As used herein, the terms “glucocorticoid-induced TNFR family relatedreceptor” or “GITR” or “GITR polypeptide” refer to GITR including, butnot limited to, native GITR, an isoform of GITR, or an interspecies GITRhomolog of GITR. GITR is a 26 kDa type I transmembrane protein. GenBank™accession numbers BC152381 and BC152386 provide exemplary human GITRnucleic acid sequences. Swiss-Prot accession number Q9Y5U5-1(TNR18_HUMAN; SEQ ID NO: 701) and GenBank™ accession number NP_004186provide exemplary human GITR amino acid sequences for isoform 1. Thisamino acid sequence is 241 amino acids in length with the first 25 aminoacid residues encoding the signal sequence. Isoform 1 is a type Imembrane protein. An exemplary mature amino acid sequence of human GITRis provided as SEQ ID NO: 700. In contrast, isoform 2 is a secreted formof human GITR and is approximately 255 amino acids in length. Swiss-Protaccession number Q9Y5U5-2 and GenBank™ accession number NP_683699provide exemplary human GITR amino acid sequences for isoform 2. Isoform3 of human GITR is approximately 234 amino acids in length. Swiss-Protaccession number Q9Y5U5-3 and GenBank™ accession number NP_683700(isoform 3 precursor) provide exemplary human GITR amino acid sequencesfor isoform 3. In a specific embodiment, the GITR is human GITR. Inanother specific embodiment, the GITR is human GITR isoform 1 (SEQ IDNO: 701). In certain embodiments, the GITR is human isoform 2 (SEQ IDNO: 702) or isoform 3 (SEQ ID NO: 703). GITR is also known as tumornecrosis factor receptor superfamily member 18 (TNFRSF18),activation-inducible TNFR family receptor (AITR), GITR-D, and CD357.Human GITR is designated GeneID: 8784 by Entrez Gene.

The amino acid sequence of an immature form of an exemplary GITR proteinfrom cynomolgus monkey is provided in SEQ ID NO: 704. The mature form ofthis exemplary protein is amino acids 26-234 of SEQ ID NO: 704.

As used herein, the terms “GITR ligand” and “GITRL” refer toglucocorticoid-induced TNFR-related protein ligand. GITRL is otherwiseknown as activation-induced TNF-related ligand (AITRL) and tumornecrosis factor ligand superfamily member 18 (TNFSF18). GenBank™accession number AF125303 provides an exemplary human GITRL nucleic acidsequence. GenBank™ accession number NP_005083 and Swiss-Prot accessionnumber Q9UNG2 provide exemplary human GITRL amino acid sequences. In aparticular embodiment, the GITRL is a human GITRL of SEQ ID NO: 716.

As used herein, the term “host cell” can be any type of cell, e.g., aprimary cell, a cell in culture, or a cell from a cell line. In specificembodiments, the term “host cell” refers to a cell transfected with anucleic acid molecule and the progeny or potential progeny of such acell. Progeny of such a cell may not be identical to the parent celltransfected with the nucleic acid molecule, e.g., due to mutations orenvironmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

As used herein, the term “effective amount” in the context of theadministration of a therapy to a subject refers to the amount of atherapy that achieves a desired prophylactic or therapeutic effect.Examples of effective amounts are provided in Section 5.4.1.3, infra.

As used herein, the terms “subject” and “patient” are usedinterchangeably. The subject can be an animal. In some embodiments, thesubject is a mammal such as a non-primate (e.g., cow, pig, horse, cat,dog, rat etc.) or a primate (e.g., monkey or human), most preferably ahuman. In certain embodiments, such terms refer to a non-human animal(e.g., a non-human animal such as a pig, horse, cow, cat or dog). Insome embodiments, such terms refer to a pet or farm animal. In specificembodiments, such terms refer to a human.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a Western Blot under non-reducing conditions showingspecificity of anti-GITR antibody 231-32-15 versus an isotype control.Antibody is blotted against human GITR recombinant protein (Hu GITRrecomb protein), mouse GITR recombinant protein (Mu GITR recombprotein), CMS5A cells expressing recombinant human GITR (CMS5A-huGITR),wild-type CMS5A cells (CMS5A-wt), protein from CD4⁺ Activated cells(CD4⁺ Activated) and protein from CD4⁺ Untreated cells (CD4⁺ Untreated).231-32-15 reactivity is seen against human GITR, recombinant human GITRin CMS5A cells, and natural human GITR in activated CD4⁺ cells.

FIGS. 2A and 2B show FACS analysis of competitive binding of theanti-GITR antibodies versus commercial (R&D Systems) anti-GITR mAb. InFIG. 2A blocking of the R&D Systems mAb is tested using the R&D mAb andthe test antibodies (antibody 1042-7, antibody 1039-45, antibody 1333-21and antibody 32-15) as indicated in the figure. The condition ‘noantibody’ shows binding of the R&D Systems mAb alone, in the absence oftest antibodies. FIG. 2B shows blocking of the anti-GITR antibody231-1039-45 using no mAb, the R&D Systems mAb and the test antibodies(antibody 1042-7, antibody 1039-45, antibody 1333-21 and antibody 32-14)as indicated in the figure. The condition ‘no antibody’ shows binding ofantibody 231-1039-45 alone, in the absence of test antibodies.

FIGS. 3A, 3B and 3C: FIG. 3A depicts staining of CMS5A-GITR byantibodies 1333-21 batch 1, 1333-21 batch 2 and R&D antibody at varyingconcentrations of antibody. FIG. 3B graphs the fluorescence intensity ofex-vivo PBMC CD3-CD19-GITR+ and CD4+CD25+GITR+ cells on staining withantibodies 1042-7, 32-15, 1039-45, 1333-21 and R&D antibody. FIG. 3Cprovides FACS analysis of CD3-CD19-GITR+ and CD4+CD25+GITR+ cells byantibody 1333-21 and the R&D Systems antibody.

FIG. 4 depicts an assessment of the costimulatory effect of anti-GITRantibody on CD4+ T cells in combination with varying concentrations ofanti-CD3 (OKT3) antibody. In the top panel the % CFSE-low cells isplotted for each antibody tested (PBS control, R&D, 1042-7, 32-15,1039-45 and 1333-21) in combination with decreasing concentrations ofOKT3 antibody (5 μg/ml, 1 μg/ml, 0.2 μg/ml, 0.04 μg/ml and 0 μg/ml). Inthe bottom panel the concentration of IFNγ (pg/ml) is plotted for eachantibody tested (PBS control, R&D, 1042-7, 32-15, 1039-45 and 1333-21)in combination with decreasing concentrations of the OKT3 antibody (5μg/ml, 1 μg/ml, 0.2 μg/ml, 0.04 μg/ml and 0 μg/ml).

FIG. 5 shows GITRL-PE binding to GITR in the presence of anti-GITRantibodies chimeric parental 231-32-15 and m6C8. A further antibodySK48E26, which recognizes IL-1β, was used as a negative control. Thepercentage of GITRL-PE binding was measured by suspension arraytechnology (Luminex® 200 system) in the presence of increasing antibodyconcentrations (12, 37, 111, 333, 1000, 3000 and 9000 ng/ml). FIG. 5shows the results from four independent repeats of this assay performedin duplicate and standard deviation was determined from n=8.

FIG. 6 is a similar graph to that shown in FIG. 5 where the percentageof GITRL-PE binding was measured by suspension array technology(Luminex® 200 system) in the presence of increasing antibodyconcentrations (12, 37, 111, 333, 1000, 3000 and 9000 ng/ml). Theanti-GITR antibodies tested were the chimeric parental 231-32-15antibody and the two humanized variants Hum231#1 and Hum231#2. Thisfigure shows the results from one experiment performed in duplicate.

FIG. 7 shows GITR ligand binding to GITR in the presence of mAbs asmeasured by surface plasmon resonance (BIAcore® T100/200). The anti-GITRantibodies tested were chimeric parental 231-32-15, humanized variantsHum231#1 and Hum231#2 and m6C8. The negative control was the anti-IL-1βantibody SK48E26.

FIGS. 8A and 8B show FACS plots of the results of a suboptimal CD3stimulation assay to assess the effects of stimulation of anti-GITRantibodies on enriched CD4⁺ T cells from two different buffy coats. FIG.8A shows the FACS analysis of cell number and proliferation of CD4 Tcells from a high responder to stimulation (buffy coat 6), whereas FIG.8B shows the FACS analysis for a low responder (buffy coat 8). Cellproliferation (CFSE; x-axis) is shown for 10 μg/ml of anti-GITR antibody(chimeric parental 231-32-15 antibody and humanized variants Hum231#1and Hum231#2). The controls used were either anti-CD3/anti-CD28 antibodyalone or no stimulation. The assay was performed in triplicate.

FIGS. 9A and 9B are histograms showing the effect of anti-GITR humanizedvariant antibodies Hum231#1 and Hum231#2 on enriched CD4 T cellproliferation (FIG. 9A) and cell number (FIG. 9B), compared to theantibody m6C8, in a suboptimal CD3 stimulation assay. The antibodieswere used at a concentration of 10 μg/ml. The end column (solid blackfill; FIGS. 9A and 9B) indicates anti-CD3/anti-CD28 simulation withoutthe addition of any anti-GITR antibodies.

FIGS. 10A, 10B, 10C and 10D show the analysis of cytokine production forIFNγ, IL-6, IL-10 and TNFα, respectively induced by the administrationof anti-GITR antibodies in a suboptimal CD3 stimulation assay. Theanti-GITR antibodies tested were chimeric parental 231-32-15 andhumanized variants Hum231#1 and Hum231#2 at concentrations of 10 μg/mland 5 μg/ml.

FIG. 11 is a histogram showing the further titration of anti-GITRantibodies and their effect on cell proliferation in a suboptimal CD3stimulation assay. The chimeric parental 231-32-15 antibody andhumanized variants Hum231#1 and Hum231#2 were used at concentrations of10 μg/ml, 5 μg/ml and 2.5 μg/ml.

FIGS. 12A and 12B show the further titration of anti-GITR antibodies andtheir effect on IFNγ production in a suboptimal CD3 stimulation assay.The chimeric parental 231-32-15 antibody and humanized variants Hum231#1and Hum231#2 were used at concentrations of 10 μg/ml, 5 μg/ml and 2.5μg/ml as plate bound (FIG. 12A) or 20 μg/ml, 10 μg/ml and 5 μg/ml assoluble antibodies (FIG. 12B).

FIG. 13 is a set of bar graphs showing the results of co-stimulationwith 5 μg/ml, plate-bound Hum231#2 on cytokine secretion by PBMCs in asuboptimal CD3 stimulation assay. The data shown in FIG. 13 are from twodonors tested on day 2 and day 4 post-stimulation. The max foldinduction over isotype control was plotted for six different cytokines(IFNγ, IL-2, TNFα, IL-10, IL-13 and IL-4). The error bars representstandard deviation for a replicate of two for each cytokine. Each donorhas been tested in at least three individual experiments.

FIGS. 14A, 14B and 14C are results of intracellular cytokine stainingassays measuring the production of IFNγ and TNFα, induced by plate-boundHum231#2, Hum231#2w or pab1989 (the IgG4 counterpart of Hum231#2w) undersuboptimal CD3 stimulation. FIG. 14A is a set of flow cytometry plotsshowing the co-staining of IFNγ and TNFα for CD4+ and CD8+ T cells. Thepercentage of IFNγ+ monofunctional T cells, TNFα+ monofunctional T cellsor IFNγ+ TNFα+ polyfunctional T cells was plotted for Hum231#2,Hum231#2w, pab1989 or isotype control over a range of suboptimalanti-CD3 antibody concentrations (FIGS. 14B and 14C). Each dot in FIGS.14B and 14C represents a replicate of two for the condition tested. Theerror bars represent standard deviation. The anti-GITR antibodies wereused at a concentration of 5 μg/ml. The graphs are representative ofexperiments using PBMCs from six (FIGS. 14A and 14B) and four (FIG. 14C)different donors, respectively.

FIGS. 15A, 15B and 15C are a set of bar graphs showing results ofexperiments comparing the anti-GITR antibody Hum231#2 under differentcross-linking conditions. FIG. 15A is a bar graph showing the maximumfold induction from isotype control for the percentage of IFNγ+ TNFα+polyfunctional CD8+ T cells using PBMCs co-stimulated by 5 μg/mlplate-bound (PB) or soluble Hum231#2 or isotype control. The error barsrepresent standard deviation. * represents p<0.05 and ** representsp<0.005 (unpaired T-test). In FIGS. 15B and 15C, the maximum foldinduction over isotype control for six different cytokines was plottedfor either plate-bound Hum231#2 (FIG. 15B) or anti-Fc cross-linkedHum231#2 (FIG. 15C). The error bars represent standard deviation from areplicate of two for each cytokine.

FIGS. 16A and 16B show the results of anti-CD3/anti-CD28 and anti-GITRantibody stimulation on T effector (T-eff) and T regulatory cells(Tregs). FIG. 16A shows that activated T-effector and T-regulatory cellsexpress GITR on their cell surface following stimulation withanti-CD3/anti-CD28 alone or in conjunction with anti-GITR antibodies.However, as is shown in FIG. 16B, costimulation with anti-GITRantibodies preferentially expands effector T-cells over T-regulatorycells. Cell expansion/proliferation (CFSE; y-axis) is shown for 10 μg/mlof anti-GITR antibodies (chimeric parental 231-32-15 antibody andhumanized variants Hum231#1 and Hum231#2) on buffy coat 8. The controlsused were either anti-CD3/anti-CD28 antibody at 125 ng/ml alone or nostimulation.

FIGS. 17A and 17B show the results on T cell proliferation by theanti-GITR antibodies tested. FIG. 17A shows the proliferation of CD4cells and FIG. 17B shows the proliferation of CD8 cells in total PBMCsstimulated with 31.25 ng/ml anti-CD3 antibody. Chimeric parental231-32-15 antibody and humanized variants Hum231#1 and Hum231#2 weretested at a concentration of 10 μg/ml.

FIGS. 18A, 18B and 18C are graphs showing the results of a GITRNF-κB-luciferase reporter assay in the absence or presence of 0.3 μg/mlof a plate-bound anti-CD3 antibody (Clone SP34). FIG. 18A is a graphshowing the luciferase relative light units (RLU) at a range ofanti-GITR antibody concentrations at 18-hour post-stimulation in thepresence of the anti-CD3 antibody. FIG. 18B is a graph showingluciferase RLU at different anti-GITR antibody concentrations at 5-hourpost-stimulation in the absence of the anti-CD3 antibody. FIG. 18C is agraph showing the highest ratios of luciferase expression (GITRAb/isotype control) at 0, 2, 5, 6, 8 and 18 hrs post-stimulation. Theerror bars represent standard deviation from duplicates. The anti-GITRantibodies tested were Hum231#2w and m6C8. The data shown arerepresentative of four experiments with anti-CD3 antibody or twoexperiments without anti-CD3 antibody.

FIG. 19A is a bar graph showing the normalized receptor density of humanGITR on activated nTregs, CD4+ T cells or CD8+ T cells as measured byflow cytometry. The anti-GITR antibody used was a PE-conjugated mouseanti-human GITR antibody (Biolegend: 621; 311604/B171072). The errorbars represent standard deviation. FIG. 19B is a graph examining theanti-GITR antibody Hum231#2w using an Fc gamma receptor IIIA (CD16)reporter cell line. Jurkat NFAT-luciferase reporter cells overexpressingCD16A with the high affinity 158 V/V polymorphism were co-cultured withactivated primary nTregs and T effector cells for 20 hours at 37° C. inthe presence of Hum231#2w or an isotype control. The relative lightunits (RLU) were recorded after 20 hours, representing CD16A binding. ARLU represents the RLU of the anti-GITR antibody minus that of theisotype control. The error bars represent standard deviation (n=2). Thedata shown are representative of experiments using cells from threedonors. FIG. 19C is a set of histograms showing the surface expressionof GITR measured by flow cytometry. Samples were collected from theblood of healthy human donors (a-c, n=3) or from tumor tissues ofnon-small cell lung cancer patients (NSCLC) (d-f, n=3). The cellpopulations were defined as: Tconv (CD3+, CD4+, CD8a−, CD25low, FOXP3−)or Treg (CD3+, CD4+, CD8a−, CD25high, FOXP3+).

FIGS. 20A, 20B and 20C are results from experiments using PBMCs fromAfrican green monkey (AGM). FIG. 20A is a set of flow cytometry plots ofthe staining of activated CD4+ and CD8+ T cells from African greenmonkey (AGM) using the anti-GITR antibody Hum231#2 and an anti-PD-1antibody. Healthy AGM PBMCs were activated with anti-CD3 antibody (cloneSP34.2) or ConA plus IL-2 (20 U/ml) for 3 days. The flow cytometry plotsare representative of experiments using PBMCs from three different AGMs.FIGS. 20B and 20C are results of a CD3 substimulation assay using AGMPBMCs. FIG. 20B is a pair of flow cytometry plots showing theco-staining of CD8 and IFNγ for cells co-stimulated by Hum231#2w orisotype control. In FIG. 20C, the percentage of IFNγ+ AGM CD8+ T cellswas plotted for different anti-GITR antibody concentrations. Each dotrepresents a replicate of two wells and the error bars representstandard deviation. The data shown in FIGS. 20B and 20C arerepresentative of experiments using PBMCs from two AGMs.

FIGS. 21A and 21B are results from the staining of surface OX40 and PD-1on CD4+ and CD8+ T cells stimulated with plate-bound 0.8 μg/ml of ananti-CD3 antibody and 5 μg/ml of the anti-GITR antibody Hum231#2. FIG.21A is a set of flow cytometry plots and histograms showing co-stainingof OX40 and PD-1. In FIG. 21B, each bar represents the MFI value forPD-1 and OX40 on CD4+ and CD8+ T cells stimulated with Hum231#2 (blackbars), isotype control (gray bars) or media only (white bars). The errorbars represent standard deviation. The flow cytometry plots and graphsare representative of experiments using PBMCs from one donor.

FIGS. 22A and 22B show the design of the mutated libraries for thegeneration of germlined antibody variants. The different framework andCDR positions included in the library based on the IGHV1-2*02 VH humangermline are shown in FIG. 22A (SEQ ID NOS 37-53, respectively, in orderof appearance) and for the library based on the IGKV4-1*01 VL humangermline in FIG. 22B (SEQ ID NOS 54-71, respectively, in order ofappearance).

FIG. 23 is a table listing 17 germlined antibody variants and detailingtheir heavy and light chain variable regions with corresponding SEQ IDnumbers. The table shows the number of extra germline amino acids andthe mean relative affinity of the variant antibodies compared to thechimeric parental 231-32-15 antibody.

FIGS. 24A-C are a table listing 107 germlined antibody variants anddetailing their heavy and light chain variable regions withcorresponding SEQ ID numbers.

FIGS. 25A and 25B show GITRL-PE binding to GITR in the presence of aselection of anti-GITR germlined antibody variants. The percentage ofGITRL-PE binding was measured by suspension array technology (Luminex®200 system) in the presence of increasing antibody concentrations (12,37, 111, 333, 1000, 3000 and 9000 ng/ml).

FIGS. 26A and 26B show the effect on cell proliferation (% CFSE Low) ofthe germlined antibody variants compared to the chimeric parental231-32-15 antibody and the humanized variants Hum231#1 and Hum231#2 onenriched CD4 T cells from two buffy coats, BC4 (FIG. 26A) and BC9 (FIG.26B). A suboptimal CD3 stimulation assay was performed using plate boundanti-CD3 antibody at 125 ng/ml with either plate bound or solubleisotype control. Anti-GITR antibodies were used at a concentration of 10μg/ml.

FIGS. 27A and 27B show the effect on cytokine release of IFNγ and IL-10,respectively of the germlined antibody variants compared to the chimericparental 231-32-15 antibody and the humanized variants Hum231#1 andHum231#2 on enriched CD4 T cells from buffy coat BC4. A suboptimal CD3stimulation assay was performed using plate bound anti-CD3 antibody at125 ng/ml with either plate bound or soluble isotype control. Anti-GITRantibodies were used at a concentration of 10 μg/ml and the cytokinelevels were measured in the culture supernatant.

FIGS. 28A and 28B show the effect on cytokine release of IFNγ and IL-10,respectively of the germlined antibody variants compared to the chimericparental 231-32-15 antibody and the humanized variants Hum231#1 andHum231#2 on enriched CD4 T cells from buffy coat BC9. A suboptimal CD3stimulation assay was performed using plate bound anti-CD3 antibody at125 ng/ml with either plate bound or soluble isotype control. Anti-GITRantibodies were used at a concentration of 10 μg/ml and the cytokinelevels were measured in the culture supernatant.

FIGS. 29A and 29B show the percentage of IFNγ positive CD4⁺ T-cells (asmeasured by intracellular staining) of germlined antibody variantscompared to the chimeric parental 231-32-15 antibody and the humanizedvariants Hum231#1 and Hum231#2 on enriched CD4 T cells from two buffycoats. FIG. 29A shows the results from buffy coat 13 (BC13) and FIG. 29Bshows the results from buffy coat 18 (BC18).

FIGS. 30A-C are a set of graphs showing the results of a GITRNF-κB-luciferase reporter assay in the presence of 0.3 μg/ml anti-CD3antibody. The anti-GITR antibodies tested in this assay were Hum231#2wand 20 germline variants: pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, pab2159, pab2160 and pab2161. InFIGS. 30A-C, the luciferase RLU at 18-hour post-stimulation was plottedfor different anti-GITR antibody concentrations tested. The error barsrepresent standard deviation. FIGS. 30D-F are a set of graphs showingthe results of a GITR NF-κB-luciferase reporter assay in the absence ofan anti-CD3 antibody. The anti-GITR antibodies tested in this assay werem6C8, Hum231#2w and 20 germline variants: pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, pab2159, pab2160and pab2161. In FIGS. 30D-F, the luciferase RLU at 6-hourpost-stimulation was plotted for different anti-GITR antibodyconcentrations tested. The error bars represent standard deviation. Thegraphs and plots are representative of data from two experiments (FIGS.30A-C) or one experiment (FIGS. 30D-F).

FIG. 31 shows the loss of binding of 1624-5 pre-B cells expressing thechimeric parental 231-32-15 antibody to biotinylated GITR (GITR-bio)when GITR-bio was pre-incubated with chimeric parental 231-32-15,Hum231#1 or Hum231#2 antibodies. FIG. 31 right-hand profile depicts thebinding of 1624-5 pre-B cells expressing the chimeric parental 231-32-15antibody to GITR-bio. In the left-hand profile however, there is loss ofbinding of 1624-5 cells expressing the chimeric parental 231-32-15antibody to GITR-bio following pre-incubation of GITR-bio with eitherthe chimeric parental 231-32-15, Hum231#1 or Hum231#2 antibodies.

FIG. 32 shows the results of an epitope competition assay measured bysurface plasmon resonance (BIAcore® T100/200). GITR antigen wasimmobilized on a CM5 sensor chip and the anti-GITR antibodies applied ata concentration of 300 nM. Chimeric parental 231-32-15 antibody wasapplied first followed by the application of the murine antibody 6C8.

FIGS. 33A and 33B are the results of an epitope mapping experiment usinga cellular library expressing GITR variants generated by error pronePCR. Shown in FIGS. 33A and 33B is an alignment of sequences from theGITR variants that bind to a polyclonal anti-GITR antibody but do notbind to the anti-GITR chimeric parental 231-32-15 antibody.

FIGS. 34A and B are the result of an epitope mapping experiment usingalanine scanning. The following positions in human GITR (numberedaccording to SEQ ID NO: 701) were separately mutated to an Alanine:P28A, T29A, G30A, G31A, P32A, T54A, T55A, R56A, C57A, C58A, R59A, D60A,Y61A, P62A, G63A, E64A, E65A, C66A, C67A, S68A, E69A, W70A, D71A, C72A,M73A, C74A, V75A and Q76A. The antibodies tested in the experiment shownin FIG. 34A included: the monoclonal anti-GITR antibodies Hum231#2,three germline variants (pab1967, pab1975 and pab1979) and the m6C8antibody; and a polyclonal anti-GITR antibody (AF689, R&D systems). FIG.34A is a table summarizing the binding of Hum231#2, three germlinevariants (pab1967, pab1975 and pab1979) and the reference antibody m6C8to 1624-5 cells expressing human GITR alanine mutants. FIG. 34B is a setof flow cytometry plots showing the staining of 1624-5 cells expressingwild type human GITR, D60A mutant, or G63A mutant using the monoclonalantibodies 231-32-15, Hum231#2, or m6C8, or a polyclonal antibody. Thepercentage of GITR positive cells is indicated in each plot.

FIG. 35A is a sequence alignment of human GITR, V1M cynomolgus GITR, andV1M/Q62P/S63G cynomolgus GITR, highlighting the positions 62 and 63where two amino acids from cynomolgus GITR (GlnSer) were replaced bycorresponding residues in human GITR (ProGly). FIG. 35B is a set of flowcytometry plots showing the staining of 1624-5 cells expressing humanGITR, V1M cynomolgus GITR, or V1M/Q62P/S63G cynomolgus GITR using themonoclonal antibodies 231-32-15, Hum231#2, or m6C8, or a polyclonalanti-GITR antibody.

5. DETAILED DESCRIPTION

Provided herein are antibodies (e.g., monoclonal antibodies), andantigen-binding fragments thereof, that specifically bind to GITR (e.g.,human GITR) and modulate GITR activity. For example, in one aspect,provided herein is an antibody(ies) or fragment(s) thereof thatspecifically binds to GITR and enhances, induces, or increases one ormore GITR activities. In a specific embodiment, the antibody(ies) orantigen-binding fragment(s) is isolated.

Also provided are isolated nucleic acids (polynucleotides), such ascomplementary DNA (cDNA), encoding such antibodies, and antigen-bindingfragments thereof. Further provided are vectors (e.g., expressionvectors) and cells (e.g., host cells) comprising nucleic acids(polynucleotides) encoding such antibodies or antigen-binding fragmentsthereof. Also provided are methods of making such antibodies. In otheraspects, provided herein are methods and uses for inducing, increasingor enhancing a GITR activity, and treating certain conditions, such ascancer and infectious diseases. Related compositions (e.g.,pharmaceutical compositions), kits, and detection methods are alsoprovided.

5.1 Antibodies

In a specific aspect, provided herein are antibodies (e.g., monoclonalantibodies, such as chimeric or humanized antibodies) and fragmentsthereof which specifically bind to GITR (e.g., human GITR). In someembodiments, an antibody or antigen-binding fragment thereof describedherein partially inhibits GITRL (e.g., human GITRL) from binding to GITR(e.g., human GITR). In certain embodiments, an antibody orantigen-binding fragment thereof described herein inhibits binding ofGITRL (e.g., human GITRL) to GITR (e.g., human GITR) by less than 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10%as assessed by an assay known to one of skill in the art or describedherein. In a specific embodiment, an antibody or antigen-bindingfragment thereof described herein inhibits binding of GITRL (e.g., humanGITRL) to GITR (e.g., human GITR) by less than 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% as assessed by theassay described in Example 2, infra (e.g., Sections 6.2.5.2 or 6.2.5.4,infra). In another specific embodiment, an antibody or antigen-bindingfragment thereof described herein at a concentration of 1000 ng/ml, 950ng/ml, 900 ng/ml, 850 ng/ml, 800 ng/ml, 750 ng/ml, 700 ng/ml, 650 ng/ml,600 ng/ml, 550 ng/ml, 500 ng/ml, 450 ng/ml, 400 ng/ml, 350 ng/ml, 333ng/ml, 300 ng/ml, 250 ng/ml, 200 ng/ml, 100 ng/ml, 50 ng/ml or 10 ng/mlinhibits binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM oflabeled GITRL (e.g., GITRL-PE) to GITR coupled to beads (e.g., humanGITR coupled to Luminex® beads) at a concentration of 9 pg/ml, 8 pg/ml,7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead by less than 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10%relative to the binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM,0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nMof labeled GITRL to the GITR coupled beads at a concentration of 9pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay (e.g., Luminex® 200 system). Inanother specific embodiment, an antibody or antigen-binding fragmentthereof described herein at concentration of 1000 ng/ml to 750 ng/ml,1000 ng/ml to 500 ng/ml, 850 ng/ml to 500 ng/ml, 750 ng/ml to 500 ng/ml,600 ng/ml to 500 ng/ml, 500 ng/ml to 400 ng/ml, 400 ng/ml to 300 ng/ml,or 300 ng/ml to 200 ng/ml inhibits binding of 1.5 nM, 1.4 nM, 1.3 nM,1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM,0.3 nM, 0.2 nM, 0.1 nM of labeled GITRL (e.g., GITRL-PE) to GITR coupledto beads (e.g., human GITR coupled to Luminex® beads) at a concentrationof 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml perbead by less than 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,30%, 25%, 20% or 10% relative to the binding of 1.5 nM, 1.4 nM, 1.3 nM,1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM,0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL to the GITR coupled beads at aconcentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or3 pg/ml per bead in the absence of the anti-GITR antibody orantigen-binding fragment thereof in a suspension array assay (e.g.,Luminex® 200 system). In another specific embodiment, an antibody orantigen-binding fragment thereof at a concentration of 1000 ng/mlinhibits less than 80% (in some embodiments, 40% to 70%, 50%, to 80%, or40% to 80%) of 0.5 nM labeled GITRL (e.g., human GITRL) from binding toGITR coupled to beads (e.g., human GITR coupled to Luminex® beads) at aconcentration of 5 pg/ml/bead relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay.

In another specific embodiment, an antibody or antigen-binding fragmentthereof described herein at concentration of 3500 ng/ml, 3400 ng/ml,3300 ng/ml, 3200 ng/ml, 3100 ng/ml, 3000 ng/ml, 2900 ng/ml, 2800 ng/ml,2700 ng/ml, 2600 ng/ml, 2500 ng/ml, 2400 ng/ml, 2300 ng/ml, 2200 ng/ml,2100 ng/ml, 2000 ng/ml, 1900 ng/ml, 1800 ng/ml, 1700 ng/ml, 1600 ng/ml,1500 ng/ml, 1400 ng/ml, 1300 ng/ml, 1200 ng/ml, or 1100 ng/ml inhibitsbinding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM,0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeledGITRL (e.g., GITRL-PE) to GITR coupled to beads (e.g., human GITRcoupled to Luminex® beads) at a concentration of 9 pg/ml, 8 pg/ml, 7pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead by less than 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10%relative to the binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM,0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nMof labeled GITRL to the GITR coupled beads at a concentration of 9pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay (e.g., Luminex® 200 system). Inanother specific embodiment, an antibody or antigen-binding fragmentthereof described herein at concentration of 3500 ng/ml to 3200 ng/ml,3500 ng/ml to 3000 ng/ml, 3200 ng/ml to 2500 ng/ml, 3000 to 2200 ng/ml,2500 ng/ml to 1800 ng/ml, 2000 ng/ml to 1500 ng/ml, 1700 ng/ml to 1200ng/ml, or 1500 ng/ml to 1000 ng/ml inhibits binding of 1.5 nM, 1.4 nM,1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM,0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL (e.g., GITRL-PE) toGITR coupled to beads (e.g., human GITR coupled to Luminex® beads) at aconcentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or3 pg/ml per bead by less than 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%,45%, 40%, 35%, 30%, 25%, 20% or 10% relative to the binding of 1.5 nM,1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM,0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL to the GITRcoupled beads at a concentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml,5 pg/ml, 4 pg/ml or 3 pg/ml per bead in the absence of the anti-GITRantibody or antigen-binding fragment thereof in a suspension array assay(e.g., Luminex® 200 system).

In a certain embodiment, an antibody or antigen-binding fragment thereofdescribed herein at a concentration of 3000 ng/ml inhibits binding of0.5 nM GITRL (e.g., human GITRL) to GITR (e.g., human GITR) by less than85% or less than 80% (in some embodiments, 60% to 85%, 60% to 80%, 70%to 85% or 70% to 80%) when GITR (e.g., human GITR) is coupled to beads(e.g., Luminex® beads) at a concentration of 5 pg/ml per bead relativeto the binding of 0.5 nM of labeled GITRL to GITR coupled beads at aconcentration of 5 pg/ml/bead in the absence of the anti-GITR antibodyor antigen-binding fragment thereof, in a suspension array assay (e.g.,Luminex® 200 system). In a certain embodiment, an antibody orantigen-binding fragment thereof described herein at a concentration of1000 ng/ml inhibits binding of 0.5 nM GITRL (e.g., human GITRL) to GITR(e.g., human GITR) by less than 85%, less than 80% or less than 75% (insome embodiments, 60% to 85%, 60% to 80%, 70% to 85% or 70% to 80%) whenGITR (e.g., human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml per bead relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof, in a suspension array assay (e.g., Luminex® 200 system). In acertain embodiment, an antibody or antigen-binding fragment thereofdescribed herein at a concentration of 333 ng/ml inhibits binding of 0.5nM GITRL (e.g., human GITRL) to GITR (e.g., human GITR) by less than 70%or less than 65% (in some embodiments, 50% to 70%, 55% to 70%, 50% to65% or 50% to 60%) when GITR (e.g., human GITR) is coupled to beads(e.g., Luminex® beads) at a concentration of 5 pg/ml per bead relativeto the binding of 0.5 nM of labeled GITRL to GITR coupled beads at aconcentration of 5 pg/ml/bead in the absence of the anti-GITR antibodyor antigen-binding fragment thereof, in a suspension array assay (e.g.,Luminex® 200 system). In a certain embodiment, an antibody orantigen-binding fragment thereof described herein at a concentration of111 ng/ml inhibits binding of 0.5 nM GITRL (e.g., human GITRL) to GITR(e.g., human GITR) by less than 65%, less than 60% or less than 55% (insome embodiments, 40% to 65%, 40% to 60%, 40% to 55% or 30% to 60%) whenGITR (e.g., human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml per bead relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof, in a suspension array assay (e.g., Luminex® 200 system). In acertain embodiment, an antibody or antigen-binding fragment thereofdescribed herein at a concentration of 37 ng/ml inhibits binding of 0.5nM GITRL (e.g., human GITRL) to GITR (e.g., human GITR) by less than 40%(in some embodiments, 20% to 40%, 20% to 30%, or 15% to 35%) when GITR(e.g., human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml per bead relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof, in a suspension array assay (e.g., Luminex® 200 system). In acertain embodiment, an antibody or antigen-binding fragment thereofdescribed herein at a concentration of 12 ng/ml inhibits binding of 0.5nM GITRL (e.g., human GITRL) to GITR (e.g., human GITR) by less than 20%(in some embodiments, 10% to 20%) when GITR (e.g., human GITR) iscoupled to beads (e.g., Luminex® beads) at a concentration of 5 pg/mlper bead relative to the binding of 0.5 nM of labeled GITRL to GITRcoupled beads at a concentration of 5 pg/ml/bead in the absence of theanti-GITR antibody or antigen-binding fragment thereof, in a suspensionarray assay (e.g., Luminex® 200 system).

In certain embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, or 75% of GITRL (e.g., human GITRL) binds toGITR (e.g., human GITR) in the presence of an antibody orantigen-binding fragment thereof described herein assessed by an assayknown to one of skill in the art or described herein. In a specificembodiment, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, or 75% of GITRL (e.g., human GITRL) binds to GITR (e.g.,human GITR) in the presence of an antibody or antigen-binding fragmentthereof described herein as assessed by the assay described in Example2, infra (e.g., Section 6.2.5.2 or 6.2.5.4, infra). In another specificembodiment, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, or 75% of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM,0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nMof labeled GITRL (e.g., labeled human GITRL, such as hGITRL-PE) binds toGITR coupled to beads (e.g., human GITR coupled to Luminex® beads) at aconcentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or3 pg/ml per bead in the presence of 1000 ng/ml, 950 ng/ml, 900 ng/ml,850 ng/ml, 800 ng/ml, 750 ng/ml, 700 ng/ml, 650 ng/ml, 600 ng/ml, 550ng/ml, 500 ng/ml, 450 ng/ml, 400 ng/ml, 350 ng/ml, 333 ng/ml, 300 ng/ml,250 ng/ml or 200 ng/ml of an antibody or antigen-binding fragmentthereof described herein relative to the binding of 1.5 nM, 1.4 nM, 1.3nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4nM, 0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL to the GITR coupled beadsat a concentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4pg/ml or 3 pg/ml per bead in the absence of the anti-GITR antibody orantigen-binding fragment thereof in a suspension array assay (e.g.,Luminex® 200 system). In another specific embodiment, at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of 1.5 nM,1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM,0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL (e.g., labeledhuman GITRL, such as hGITRL-PE) binds to GITR coupled to beads (e.g.,human GITR coupled to Luminex® beads) at a concentration of 9 pg/ml, 8pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead in thepresence of 1000 ng/ml to 900 ng/ml, 1000 ng/ml to 850 ng/ml, 900 ng/mlto 800 ng/ml, or 850 ng/ml to 750 ng/ml, or 800 to 750 ng/ml relative tothe binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeledGITRL to the GITR coupled beads at a concentration of 9 pg/ml, 8 pg/ml,7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead in the absence ofthe anti-GITR antibody or antigen-binding fragment thereof in asuspension array assay (e.g., Luminex® 200 system). In another specificembodiment, at least 20%, at least 25% or at least 30% of 0.5 nM oflabeled GITRL (e.g., labeled human GITRL, such as hGITRL-PE) binds toGITR coupled to beads (e.g., human GITR coupled to Luminex® beads) at aconcentration of 5 pg/ml/bead in the presence of 1000 ng/ml of anantibody or antigen-binding fragment thereof relative to the binding of0.5 nM of labeled GITRL to GITR coupled beads at a concentration of 5pg/ml/bead in the absence of the anti-GITR antibody or antigen-bindingfragment thereof in a suspension array assay.

In another specific embodiment, at least 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of 1.5 nM, 1.4 nM, 1.3 nM, 1.2nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3nM, 0.2 nM, 0.1 nM of labeled GITRL (e.g., labeled human GITRL, such ashGITRL-PE) binds to GITR coupled to beads (e.g., human GITR coupled toLuminex® beads) at a concentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead in the presence of 3500ng/ml, 3400 ng/ml, 3300 ng/ml, 3200 ng/ml, 3100 ng/ml, 3000 ng/ml, 2900ng/ml, 2800 ng/ml, 2700 ng/ml, 2600 ng/ml, 2500 ng/ml, 2400 ng/ml, 2300ng/ml, 2200 ng/ml, 2100 ng/ml, 2000 ng/ml, 1900 ng/ml, 1800 ng/ml, 1700ng/ml, 1600 ng/ml, 1500 ng/ml, 1400 ng/ml, 1300 ng/ml, 1200 ng/ml, 1100ng/ml or 1000 ng/ml of an antibody or antigen-binding fragment thereofdescribed herein relative to the binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3nM, 0.2 nM or 0.1 nM of labeled GITRL to the GITR coupled beads at aconcentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or3 pg/ml per bead in the absence of the anti-GITR antibody orantigen-binding fragment thereof in a suspension array assay (e.g.,Luminex® 200 system). In another specific embodiment, at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of 1.5 nM,1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM,0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL (e.g., labeledhuman GITRL, such as hGITRL-PE) binds to GITR coupled to beads (e.g.,human GITR coupled to Luminex® beads) at a concentration of 9 pg/ml, 8pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead in thepresence of 3500 ng/ml to 3200 ng/ml, 3500 ng/ml to 3000 ng/ml, 3200ng/ml to 2500 ng/ml, 3000 to 2200 ng/ml, 2500 ng/ml to 1800 ng/ml, 2000ng/ml to 1500 ng/ml, 1700 ng/ml to 1200 ng/ml, or 1500 ng/ml to 1000ng/ml of an antibody or antigen-binding fragment thereof describedherein relative to the binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nMor 0.1 nM of labeled GITRL to the GITR coupled beads at a concentrationof 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml perbead in the absence of the anti-GITR antibody or antigen-bindingfragment thereof in a suspension array assay (e.g., Luminex® 200system).

In another specific embodiment, at least 20%, at least 25% or at least30% of 0.5 nM of labeled GITRL (e.g., labeled human GITRL, such ashGITRL-PE) binds to GITR coupled to beads (e.g., human GITR coupled toLuminex® beads) at a concentration of 5 pg/ml/bead in the presence of3000 ng/ml of an antibody or antigen-binding fragment thereof relativeto the binding of 0.5 nM of labeled GITRL to GITR coupled beads at aconcentration of 5 pg/ml/bead in the absence of the anti-GITR antibodyor antigen-binding fragment thereof in a suspension array assay. Inanother specific embodiment, at least 25%, at least 30%, at least 40%,or at least 50% (in some embodiments, 25% to 60%, 40% to 60%, 40% to70%, or 25% to 50%) of 0.5 nM of labeled GITRL (e.g., labeled humanGITRL, such as hGITRL-PE) binds to GITR coupled to beads (e.g., humanGITR coupled to Luminex® beads) at a concentration of 5 pg/ml/bead inthe presence of 1000 ng/ml of an antibody or antigen-binding fragmentthereof relative to the binding of 0.5 nM of labeled GITRL to GITRcoupled beads at a concentration of 5 pg/ml/bead in the absence of theanti-GITR antibody or antigen-binding fragment thereof in a suspensionarray assay. In another specific embodiment, at least 30%, at least 40%,at least 50% or at least 60% (in some embodiments, 30% to 60%, 40% to60%, 40% to 70%, or 30% to 50%) of 0.5 nM of labeled GITRL (e.g.,labeled human GITRL, such as hGITRL-PE) binds to GITR coupled to beads(e.g., human GITR coupled to Luminex® beads) at a concentration of 5pg/ml/bead in the presence of 333 ng/ml of an antibody orantigen-binding fragment thereof relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay. In another specific embodiment, atleast 40%, at least 50%, at least 60% or at least 65% (in someembodiments, 40% to 70%, 40% to 60%, 40% to 65%, or 40% to 50%) of 0.5nM of labeled GITRL (e.g., labeled human GITRL, such as hGITRL-PE) bindsto GITR coupled to beads (e.g., human GITR coupled to Luminex® beads) ata concentration of 5 pg/ml/bead in the presence of 111 ng/ml of anantibody or antigen-binding fragment thereof relative to the binding of0.5 nM of labeled GITRL to GITR coupled beads at a concentration of 5pg/ml/bead in the absence of the anti-GITR antibody or antigen-bindingfragment thereof in a suspension array assay. In another specificembodiment, at least 60%, at least 70% or at least 80% (in someembodiments 60% to 80%, 70% to 80% or 75% to 85%) of 0.5 nM of labeledGITRL (e.g., labeled human GITRL, such as hGITRL-PE) binds to GITRcoupled to beads (e.g., human GITR coupled to Luminex® beads) at aconcentration of 5 pg/ml/bead in the presence of 37 ng/ml of an antibodyor antigen-binding fragment thereof relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay. In another specific embodiment, atleast 80%, at least 85% or at least 90% (in some embodiments 80% to 90%or 85% to 95%) of 0.5 nM of labeled GITRL (e.g., labeled human GITRL,such as hGITRL-PE) binds to GITR coupled to beads (e.g., human GITRcoupled to Luminex® beads) at a concentration of 5 pg/ml/bead in thepresence of 12 ng/ml of an antibody or antigen-binding fragment thereofrelative to the binding of 0.5 nM of labeled GITRL to GITR coupled beadsat a concentration of 5 pg/ml/bead in the absence of the anti-GITRantibody or antigen-binding fragment thereof in a suspension array assay

In a certain embodiment, an antibody or antigen-binding fragment thereofdescribed herein at a concentration of 3000 ng/ml does not inhibitbinding of 0.5 nM GITRL (e.g., human GITRL) to GITR (e.g., human GITR)by more than 15% or more than 20% when GITR (e.g., human GITR) iscoupled to beads (e.g., Luminex® beads) at a concentration of 5 pg/mlper bead relative to the binding of 0.5 nM of labeled GITRL to GITRcoupled beads at a concentration of 5 pg/ml/bead in the absence of theanti-GITR antibody or antigen-binding fragment thereof, in a suspensionarray assay (e.g., Luminex® 200 system). In a certain embodiment, anantibody or antigen-binding fragment thereof described herein at aconcentration of 1000 ng/ml does not inhibit binding of 0.5 nM GITRL(e.g., human GITRL) to GITR (e.g., human GITR) by more than 15%, morethan 20% or more than 25% when GITR (e.g., human GITR) is coupled tobeads (e.g., Luminex® beads) at a concentration of 5 pg/ml per beadrelative to the binding of 0.5 nM of labeled GITRL to GITR coupled beadsat a concentration of 5 pg/ml/bead in the absence of the anti-GITRantibody or antigen-binding fragment thereof, in a suspension arrayassay (e.g., Luminex® 200 system). In a certain embodiment, an antibodyor antigen-binding fragment thereof described herein at a concentrationof 333 ng/ml does not inhibit binding of 0.5 nM GITRL (e.g., humanGITRL) to GITR (e.g., human GITR) by more than 30% or more than 35% whenGITR (e.g., human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml per bead relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof, in a suspension array assay (e.g., Luminex® 200 system). In acertain embodiment, an antibody or antigen-binding fragment thereofdescribed herein at a concentration of 111 ng/ml does not inhibitbinding of 0.5 nM GITRL (e.g., human GITRL) to GITR (e.g., human GITR)by more than 35%, more than 40% or more than 45% when GITR (e.g., humanGITR) is coupled to beads (e.g., Luminex® beads) at a concentration of 5pg/ml per bead relative to the binding of 0.5 nM of labeled GITRL toGITR coupled beads at a concentration of 5 pg/ml/bead in the absence ofthe anti-GITR antibody or antigen-binding fragment thereof, in asuspension array assay (e.g., Luminex® 200 system). In a certainembodiment, an antibody or antigen-binding fragment thereof describedherein at a concentration of 37 ng/ml does not inhibit binding of 0.5 nMGITRL (e.g., human GITRL) to GITR (e.g., human GITR) by more than 60%when GITR (e.g., human GITR) is coupled to beads (e.g., Luminex® beads)at a concentration of 5 pg/ml per bead relative to the binding of 0.5 nMof labeled GITRL to GITR coupled beads at a concentration of 5pg/ml/bead in the absence of the anti-GITR antibody or antigen-bindingfragment thereof, in a suspension array assay (e.g., Luminex® 200system). In a certain embodiment, an antibody or antigen-bindingfragment thereof described herein at a concentration of 12 ng/ml doesnot inhibit binding of 0.5 nM GITRL (e.g., human GITRL) to GITR (e.g.,human GITR) by more than 80% when GITR (e.g., human GITR) is coupled tobeads (e.g., Luminex® beads) at a concentration of 5 pg/ml per beadrelative to the binding of 0.5 nM of labeled GITRL to GITR coupled beadsat a concentration of 5 pg/ml/bead in the absence of the anti-GITRantibody or antigen-binding fragment thereof, in a suspension arrayassay (e.g., Luminex® 200 system).

In another embodiment, a certain amount of labeled GITRL (e.g., humanGITRL-PE) binds to GITR coupled to beads (e.g., human GITR coupled toLuminex® beads) in the presence of an antibody or antigen-bindingfragment thereof described herein in a method comprising: (a) couplingGITR (e.g., human GITR) to beads at a concentration of approximately 9pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead;(b) incubating the GITR coupled beads at a concentration ofapproximately 30 beads/μl, 40 beads/μl, or 50 beads/μl with 3000 ng/ml,2500 ng/ml, 2000 ng/ml, 1500 ng/ml, 1000 ng/ml, 750 ng/ml, 500 ng/ml,250 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml or 10 ng/ml of an antibody oran antigen-binding fragment thereof described herein in a well for afirst period of time (e.g., 30 minutes, 60 minutes, 1.5 hours, 2 hours,2.5 hours or 3 hours); (c) adding labeled GITRL (e.g., human GITRL-PE)to the well to obtain a final concentration of approximately 1.5 nM, 1nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or0.1 nM of the labeled GITRL and approximately 15 beads/μl, 20 beads/μl,or 25 beads/μl, and incubating for a second period of time (e.g., 30minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours or 3 hours); and (d)detecting the labeled GITRL bound to the GITR coupled beads in, e.g., asuspension array assay such as the Luminex® 200 system. In specificembodiments, the amount of the labeled GITRL bound to the GITR coupledbeads in the presence of the anti-GITR antibody or antigen-bindingfragment thereof is determined relative to the amount of labeled GITRLbound to the GITR coupled beads in the absence of the anti-GITR antibodyor antigen-binding fragment thereof. In certain embodiments, the absenceof the anti-GITR antibody or antigen-binding fragment thereof means thatno antibody or antigen-binding fragment thereof is present in the well.In other embodiments, the absence of the anti-GITR antibody orantigen-binding fragment thereof means that an isotype control antibodythat does not bind to GITR is present in the well. In accordance withthese embodiments, the amount of labeled GITRL bound to the GITR coupledbeads in the presence of the anti-GITR antibody or antigen-bindingfragment thereof is determined to be, in some embodiments, at least 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% or 15% to 60%, 20% to 60%,30% to 70%, or 20% to 50% of the amount of the labeled GITRL bound tothe GITR coupled beads in the absence of the anti-GITR antibody orantigen-binding fragment thereof.

In another embodiment, a certain amount of labeled GITRL (e.g., humanGITRL-PE) binds to GITR coupled to beads (e.g., human GITR coupled toLuminex® beads) in the presence of an antibody or antigen-bindingfragment thereof described herein in a method comprising: (a) couplingGITR (e.g., human GITR) to beads at a concentration of approximately 5pg/ml per bead; (b) incubating the GITR coupled beads at a concentrationof approximately 40 beads/μl with 3000 ng/ml, 2500 ng/ml, 2000 ng/ml,1500 ng/ml, 1000 ng/ml, 750 ng/ml, 500 ng/ml, 250 ng/ml, 100 ng/ml, 50ng/ml or 10 ng/ml of an antibody or an antigen-binding fragment thereofdescribed herein in a well for a first period of time (e.g., 30 minutes,60 minutes, 1.5 hours, 2 hours, 2.5 hours or 3 hours); (c) addinglabeled GITRL (e.g., human GITRL-PE) to the well to obtain a finalconcentration of 0.5 nM of the labeled GITRL and approximately 20beads/μl, and incubating for a second period of time (e.g., 30 minutes,1 hour, 1.5 hours, 2 hours, 2.5 hours or 3 hours); and (d) detecting thelabeled GITRL bound to the GITR coupled beads in, e.g., a suspensionarray assay such as the Luminex® 200 system. In specific embodiments,the amount of the labeled GITRL bound to the GITR coupled beads in thepresence of the anti-GITR antibody or antigen-binding fragment thereofis determined relative to the amount of labeled GITRL bound to the GITRcoupled beads in the absence of the anti-GITR antibody orantigen-binding fragment thereof. In certain embodiments, the absence ofthe anti-GITR antibody or antigen-binding fragment thereof means that noantibody or antigen-binding fragment thereof is present in the well. Inother embodiments, the absence of the anti-GITR antibody orantigen-binding fragment thereof means that an isotype control antibodythat does not bind to GITR is present in the well. In accordance withthese embodiments, the amount of labeled GITRL bound to the GITR coupledbeads in the presence of the anti-GITR antibody or antigen-bindingfragment thereof is determined to be, in some embodiments, at least 20%,25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% or 20 to 70%, 20% to 60%, 30%to 70%, or 20% to 50% of the amount of the labeled GITRL bound to theGITR coupled beads in the absence of the anti-GITR antibody orantigen-binding fragment thereof.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein at a concentration of 150 nM, 145 nM, 140 nM, 135 nM,130 nM, 125 nM, 120 nM, 115 nM, 110 nM, 105 nM or 100 nM bound to GITR(e.g., human GITR) immobilized on a chip (e.g., CM5 sensor chip)inhibits binding of 150 nM, 145 nM, 140 nM, 135 nM, 130 nM, 125 nM, 120nM, 115 nM, 110 nM, 105 nM or 100 nM of GITRL (e.g., non-covalentlylinked trimer of human GITRL) to the GITR immobilized on the chip byless than 60%, less than 55%, less than 50%, less than 45%, less than40%, less than 35%, less than 30%, less than 25%, less than 20% or lessthan 15%. In certain embodiments, an antibody or antigen-bindingfragment thereof described herein at a concentration of 125 nM bound toGITR (e.g., human GITR) immobilized on a chip (e.g., CM5 sensor chip)inhibits binding of 125 nM of GITRL (e.g., non-covalently linked trimerof human GITRL) to the GITR immobilized on the chip by less than 60%,less than 55%, less than 50%, less than 45%, less than 40%, less than35%, less than 30%, less than 25%, less than 20% or less than 15%.

In certain embodiments, an antibody or fragment thereof described hereinbinds to GITR (e.g., human GITR) with a dissociation rate constant(k_(off)) of 8.5×10⁻³ s⁻¹ or less, 3.5×10⁻³ s⁻¹ or less, 5×10⁻³ s⁻¹ orless, 2.5×10⁻³ s⁻¹ or less, 1×10⁻³ s⁻¹ or less, 8.5×10⁻⁴ s⁻¹ or less,5×10⁻⁴ s⁻¹ or less, 3.5×10⁻⁴ s⁻¹ or less, 2.5×10⁻⁴ s⁻¹ or less, 1×10⁻⁴s⁻¹ or less, 8.5×10⁻⁵⁻ s⁻¹ or less, 3.5×10⁻⁵⁻ s⁻¹ or less, 5×10⁻⁵⁻ s⁻¹or less, 2.5×10⁻⁵⁻ s⁻¹ or less, 1×10⁻⁵⁻ s⁻¹ or less, 8.5×10⁻⁶⁻ s⁻¹ orless, 5×10⁻⁶⁻ s⁻¹ or less, 3.5×10⁻⁶⁻ s⁻¹ or less, 2.5×10⁻⁶⁻ s⁻¹ or less,1×10⁻⁶⁻ s⁻¹ or less, 8.5×10⁻⁷⁻ s⁻¹ or less, 5×10⁻⁷⁻ s⁻¹ or less,2.5×10⁻⁷⁻ s⁻¹ or less, 1×10⁻⁷⁻ s⁻¹ or less, 8.5×10⁻⁸⁻ s⁻¹ or less,5×10⁻⁸⁻ s⁻¹ or less, 2.5×10⁻⁸⁻ s⁻¹ or less, 1×10⁻⁸⁻ s⁻¹ or less,8.5×10⁻⁹⁻ s⁻¹ or less, 5×10⁻⁹ s⁻¹ or less, 2.5×10⁻⁹⁻ s⁻¹ or less, or1×10⁻⁹⁻ s⁻¹ or less. In some embodiments, an antibody or fragmentthereof described herein binds to GITR (e.g., human GITR) with a k_(off)of between 9.5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁹⁻ s⁻¹, 8.5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁹⁻ s⁻¹,5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁹⁻ s⁻¹, 9.5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁸⁻ s⁻¹, 5×10⁻⁵ s⁻¹ to1×10⁻⁸⁻ s⁻¹, 9.5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁷⁻ s⁻¹, 5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁷⁻ s⁻¹,9.5×10⁻⁵⁻ s⁻¹ to 5×10⁻⁶⁻ s⁻¹, 9.5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁵⁻ s⁻¹, 8.5×10⁻³ s⁻¹to 1×10⁻⁴ s⁻¹, 5×10⁻³ s⁻¹ to 2.5×10⁻⁴ s⁻¹, 8.5×10⁻³ s⁻¹, to 1×10⁻⁵ s⁻¹,8.5×10⁻⁵⁻ s⁻¹ to 5×10⁻⁵⁻ s⁻¹. In certain embodiments, the k_(off\) isdetermined using a monovalent antibody, such as a Fab fragment, asmeasured by, e.g., BIAcore® surface plasmon resonance technology. Inother embodiments, the k_(off) is determined using a bivalent antibodyas measured by, e.g., BIAcore® surface plasmon resonance technology. Ina particular embodiment, the k_(off) is determined using an assaydescribed in Section 6, infra.

In certain embodiments, an antibody or fragment thereof described hereinbinds to GITR (e.g., human GITR) with an association rate constant(k_(on)) of at least 10⁵ M⁻¹s⁻¹, at least 2.5×10⁵ M⁻¹s⁻¹, at least3.5×10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻¹s⁻¹, at least 10⁶ M⁻¹s⁻¹, at least2.5×10⁶ M⁻¹s⁻¹, at least 3.5×10⁶ M⁻¹s⁻¹, at least 5×10⁶ M⁻¹s⁻¹, at least10⁷ M⁻¹s⁻¹, at least 5×10⁷ M⁻¹s⁻¹, at least 10⁸ M⁻¹s⁻¹, at least 5 10⁸M⁻¹s⁻¹ or at least 10⁹ M⁻¹s⁻¹. In some embodiments, an antibody orfragment thereof described herein binds to GITR (e.g., human GITR) witha k_(on) of between 1×10⁵ M⁻¹s⁻¹ to 5×10⁵ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹ to 1×10⁶M⁻¹s⁻¹, 3.5×10⁵ M⁻¹s⁻¹ to 2.5×10⁶ M⁻¹s⁻¹, 3.5×10⁵ M⁻¹s⁻¹ to 3.5×10⁶M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹ to 5×10⁶ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹ to 1×10⁷ M⁻¹s⁻¹,1×10⁵ M⁻¹s⁻¹ to 5×10⁷ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹ to 10⁸ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹to 1×10⁹ M⁻¹s⁻¹, 1×10⁶ M⁻¹s⁻¹ to 1×10⁷ M⁻¹s⁻¹, 1×10⁶ M⁻¹s⁻¹ to 1×10⁸M⁻¹s⁻¹, 1×10⁶ M⁻¹s⁻¹ to 1×10⁹ M⁻¹s⁻¹, 1×10⁷ M⁻¹s⁻¹ to 1×10⁸ M⁻¹s⁻¹,1×10⁷ M⁻¹s⁻¹ to 1×10⁹ M⁻¹s⁻¹, 1×10⁸ M⁻¹s⁻¹ to 1×10⁹ M⁻¹s⁻¹. In certainembodiments, the k_(on) is determined using a monovalent antibody, suchas a Fab fragment, as measured by, e.g., BIAcore® surface plasmonresonance technology. In other embodiments, the k_(on) is determinedusing a bivalent antibody as measured by, e.g., BIAcore® surface plasmonresonance technology. In a particular embodiment, the k_(on) isdetermined using an assay described in Section 6, infra.

In certain embodiments, an antibody or fragment thereof described hereinbinds to GITR (e.g., human GITR) with a K_(D) of less than 7 nM, 6 nM, 5nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.75 nM, 0.5nM, 0.25 nM, or 0.1 nM. In some embodiments, an antibody or fragmentthereof described herein binds to GITR (e.g., human GITR) with a K_(D)of about 7 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5nM, 1 nM, 0.75 nM, 0.5 nM, 0.25 nM, or 0.1 nM. In certain embodiments,an antibody or fragment thereof described herein binds to GITR (e.g.,human GITR) with a K_(D) of 7 nM to 2 nM, 5 nM to 3 nM, 5 nM to 1 nM, 4nM to 3 nM, 4 nM to 2 nM, 3 nM to 2 nM, 3 nM to 1 nM, 2 nM to 1 nM, 3 nMto 0.1 nM, 2 nM to 0.1 nM, 1 nM to 0.1 nM, or 0.5 nM to 0.1 nM. Incertain embodiments, the K_(D) is calculated as the quotient ofk_(off)/k_(on), and the k_(on) and k_(off) are determined using amonovalent antibody, such as a Fab fragment, as measured by, e.g.,BIAcore® surface plasmon resonance technology. In other embodiments, theK_(D) is calculated as the quotient of k_(off)/k_(on), and the k_(on)and k_(off) are determined using a bivalent antibody, such as a Fabfragment, as measured by, e.g., BIAcore® surface plasmon resonancetechnology. In a specific embodiment, the K_(D) is determined as setforth in the Examples in Section 6, infra (e.g., Example 2).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprises alight chain variable region (VL) comprising:

(a) a VL CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence KSSQSX₁X₂X₃X₄X₅X₆X₇KX₈YLX₉ (SEQ ID NO: 4),wherein:

X₁ is L, A, V, I, P, F or M

X₂ is L, A, V, I, P, F, M or S

X₃ is N, G, Q, S, T, C, W, Y or A

X₄ is S, G, N, Q, T, C, W, Y or A

X₅ is G, N, Q, S, T, C, W, Y or A

X₆ is N, G, Q, S, T, C, W, Y or A

X₇ is Q, G, N, S, T, C, W, Y or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is T, G, N, Q, S, C, W, Y, V, I or A; and/or

(b) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁ASTRX₂X₃(SEQ ID NO: 5), wherein:

X₁ is W, G, N, Q, S, T, C, Y, F, H or A

X₂ is E, D or A

X₃ is S, G, N, Q, T, C, W, Y or A; and/or

(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence QX₁X₂YX₃X₄PYT (SEQ ID NO: 6), wherein:

X₁ is N, G, Q, S, T, C, W or Y

X₂ is D, E or Y

X₃ is S, G, N, Q, T, C, W, Y or A

X₄ is Y, G, N, Q, S, T, C, W, F, H, L, or A.

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two, or all three of the VL CDRs above. Incertain embodiments, the antibody or antigen-binding fragment thereofcomprises the VL CDR1 of one of the antibodies in Table 1. In someembodiments, the antibody or antigen-binding fragment thereof comprisesthe VL CDR2 of one of the antibodies in Table 1. In certain embodiments,the antibody or antigen-binding fragment thereof comprises the VL CDR3of one of the antibodies in Table 1. In certain embodiments, theantibody or antigen-binding fragment thereof comprises one, two or allthree of the VL CDRs of one of the antibodies in Table 1 (e.g., the VLCDRs in one row of Table 1, for example, all of the VL CDRs are fromantibody 231-32-15). In some embodiments, the antibody orantigen-binding fragment thereof comprises the VL framework regionsdescribed herein. In specific embodiments, the antibody orantigen-binding fragment thereof comprises the VL framework regions(FRs) of an antibody set forth in Table 3 (e.g., one, two, three, orfour of the framework regions in one row of Table 3).

In another embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR), comprises a heavy chain variable region (VH)comprising:

(a) a VH CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁YX₂MX₃ (SEQ ID NO: 1), wherein

X₁ is D, E, G or A

X₂ is A, V, L, I, P, F, M or Y

X₃ is Y, G, N, Q, S, T, C, W, F or H; and/or

(b) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁IX₂X₃X₄SGX₅X₆X₇YX₈QKFX₉X₁₀ (SEQ ID NO: 2),wherein

X₁ is V, A, L, I, P, F, M or T

X₂ is R, K, H, Q or A

X₃ is T, G, N, Q, S, C, W, Y, V, I or P

X₄ is Y, G, N, Q, S, T, C, W, F, H, or A

X₅ is D, E, G or A

X₆ is V, A, L, I, P, F, M or T

X₇ is T, G, N, Q, S, C, W, Y, V, I, P or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is K, R, H, Q or A

X₁₀ is D, E, G or A; and/or

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence SGTVRGX₁X₂X₃ (SEQ ID NO: 3), wherein

X₁ is F, A, V, L, I, P, M, Y, W, H or S

X₂ is A, or D

X₃ is Y, G, N, Q, S, T, C, W, F, H or V.

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two or all three of the VH CDRs above. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesthe VH CDR1 of one of the antibodies in Table 2. In some embodiments,the antibody or antigen-binding fragment thereof comprises the VH CDR2of one of the antibodies in Table 2. In certain embodiments, theantibody or antigen-binding fragment thereof comprises the VH CDR3 ofone of the antibodies in Table 2. In some embodiments, the antibody orantigen-binding fragment thereof comprises one, two or all three of VHCDRs of one of the antibodies in Table 2 (e.g., the VH CDRs in one rowof Table 2, for example, all of the VH CDRs are from the antibody231-32-15). In some embodiments, the antibody or antigen-bindingfragment thereof comprises the VH frameworks described herein. Inspecific embodiments, the antibody or antigen-binding fragment thereofcomprises the VH framework regions of an antibody set forth in Table 4(e.g., one, two, three or four of the framework regions in one row ofTable 4).

TABLE 1 VL CDR Amino Acid Sequences¹ VL CDR2 VL CDR3 AntibodyVL CDR1 (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 231-32-15KSSQSLLNSGNQKNYLT (16) WASTRES (17) QNDYSYPYT (18) Hum231#1KSSQSLLNSGNQKNYLT (16) WASTRES (17) QNDYSYPYT (18) Hum231#2KSSQSLLNSGNQKNYLT (16) WASTRES (17) QNDYSYPYT (18) pab1964KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSYPYT (106) pab1965KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab1966KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab1967KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNEYSFPYT (108) pab1968KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) pab1969KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109) pab1970KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109) pab1971KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107) pab1972KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107) pab1973KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107) pab1975KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab1976KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) pab1977KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107) pab1979KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab1980KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) pab1981KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107) pab1983KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab2159KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109) pab2160KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab2161KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)   1KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)   2KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSFPYT (108)   3KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)   4KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)   5KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNEYSFPYT (108)   6KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)   7KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNEYSFPYT (108)   8KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSYPYT (106)   9KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  10KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  11KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSFPYT (109)  12KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  13KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  14KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  15KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  16KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  17KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSFPYT (108)  18KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  19KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSFPYT (109)  20KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  21KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  22KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDHSFPYT (191)  23KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSSPYT (192)  24KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  25KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  26KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSFPYT (108)  27KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSFPYT (109)  28KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  29KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  30KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  31KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  32KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  33KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSYPYT (106)  34KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  35KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  36KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  37KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSYPYT (106)  38KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  39KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNEYSFPYT (108)  40KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSYPYT (106)  41KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  42KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  43KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  44KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSFPYT (108)  45KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  46KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  47KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  48KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSFPYT (108)  49KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  50KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  51KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSSPYT (192)  52KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  53KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  54KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  55KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  56KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  57KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  58KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  59KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  60KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSYPYT (106)  61KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  62KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSFPYT (108)  63KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  64KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNEYSYPYT (106)  65KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNEYSFPYT (108)  66KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSFPYT (109)  67KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  68KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  69KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  70KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  71KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  72KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSFPYT (108)  73KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  74KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  75KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  76KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  77KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  78KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  79KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  80KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  81KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  82KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  83KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  84KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  85KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  86KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  87KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  88KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  89KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  90KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSSPYT (193)  91KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSFPYT (108)  92KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  93KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  94KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  95KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  96KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  97KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSFPYT (108)  98KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  99KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109) 100KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSSPYT (192) 101KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109) 102KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107) 103KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSSPYT (192) 104KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSYPYT (106) 105KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) 106KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107) 107KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) ¹The VL CDRs inTable 1 are determined according to Kabat.

TABLE 2 VH CDR Amino Acid Sequences² VH CDR1 VH CDR3 Antibody(SEQ ID NO:) VH CDR2 (SEQ ID NO:) (SEQ ID NO:) 231-32-15 DYAMY (13)VIRTYSGDVTYNQKFKD (14) SGTVRGFAY (15) Hum231#l DYAMY (13)VIRTYSGDVTYNQKFKD (14) SGTVRGFAY (15) Hum231#2 DYAMY (13)VIRTYSGDVTYNQKFKD (14) SGTVRGFAY (15) pabl964 GYAMY (19)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34) pabl965 GYAMY (19)VIRTFSGDVTYNQKFRG (25) SGTVRGFAY (34) pabl966 GYAMY (19)VIKTYSGGVTYNQKFRG (26) SGTVRGFAY (34) pabl967 GYAMH (20)LIRTYSGGVSYNQKFRE (27) SGTVRGFAY (34) pabl968 DYAMY (21)VIRTFSGDLTYNQKFQD (28) SGTVRGFAY (34) pabl969 EYAMH (22)LIRTYSGGVSYNQKFQG (29) SGTVRGFAY (34) pabl970 DYAMY (21)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34) pabl971 DYAMY (21)VIRTYSGDVSYNQKFRG (177) SGTVRGFAY (34) pabl972 EYAMY (23)LIRTYSGGVSYNQKFRD (31) SGTVRGFAY (34) pabl973 GYAMY (19)VIRTFSGGVTYNQKFRG (32) SGTVRGFAY (34) pabl975 EYAMH (22)LIRTYSGGVSYNQKFQG (29) SGTVRGFAY (34) pabl976 EYAMH (22)LIRTYSGGVSYNQKFQG (29) SGTVRGFAY (34) pabl977 EYAMH (22)LIRTYSGGVSYNQKFQG (29) SGTVRGFAY (34) pabl979 EYAMH (22)VIRTYSGGVSYNQKFQE (33) SGTVRGFAY (34) pabl980 EYAMH (22)VIRTYSGGVSYNQKFQE (33) SGTVRGFAY (34) pabl981 EYAMH (22)VIRTYSGGVSYNQKFQE (33) SGTVRGFAY (34) pabl983 GYAMY (19)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34) pab2159 GYAMY (19)LIRTYSGEVSYNQKFRG (144) SGTVRGFAY (34) pab2160 GYVMH (119)VIRTFSGDVSYNQKFRE (162) SGTVRGFAY (34) pab2161 EYAMH (22)LIQTYSGDVSYNQKFRG (121) SGTVRGFAY (34)   1 EYAMY (23)VIRTYSGGVTYNQKFQG (187) SGTVRGFAY (34)   2 EYAMH (22)LIRTYSGGVSYNQKFRG (148) SGTVRGFAY (34)   3 GYVMH (119)VIRTYSGEVSYNQKFQE (181) SGTVRGFAY (34)   4 EYAMY (23)LIRTFSGDVSYNQKFQD (124) SGTVRGFAY (34)   5 EYAMH (22)LIRTYSGGVTYNQKFRG (151) SGTVRGFAY (34)   6 EYAMY (23)LIRTFSGGVSYNQKFKG (135) SGTVRGFAY (34)   7 GYAMH (20)LIRTFSGGLSYNQKFRE (132) SGTVRGFAY (34)   8 GYVMY (116)VIKTFSGGVSYNQKFQE (152) SGTVRGFAY (34)   9 GYAMY (19)LIRTYSGEVSYNQKFRG (144) SGTVRGFAY (34)  10 EYAMY (23)LIRTYSGGVSYNQKFRG (148) SGTVRGFAY (34)  11 DYAMH (117)LIRTYSGGVSYNQKFRG (148) SGTVRGFAY (34)  12 GYAMY (19)VIRTFSGEVSYNQKFKG (164) SGTVRGFAY (34)  13 GYAMY (19)LIRTFSGDVTYNQKFRG (127) SGTVRGFAY (34)  14 GYVMH (119)LIRTYSGDVSYNQKRFD (146) SGTVRGFAY (34)  15 DYAMY (21)VIRTFSGDVSYNQKFRE (162) SGTVRGFAY (34)  16 GYAMY (19)LIRTFSGGVTYNQKFRE (140) SGTVRGFAY (34)  17 EYAMY (23)VIQTFSGGVTYNQKFRG (157) SGTVRGFAY (34)  18 GYAMY (19)LIRTFSGEVTYNQKFRG (130) SGTVRGFAY (34)  19 GYAMY (19)LIRTYSGGLSYNQKFQD (145) SGTVRGFAY (34)  20 DYAMY (21)VIRTFSGDLSYNQKFRG (114) SGTVRGFAY (34)  21 GYVMH (119)VIRTFSGDVSYNQKFRE (162) SGTVRGFAY (34)  22 GYAMY (19)VIRTFSGDVTYNQKFRG (25) SGTVRGFAY (34)  23 GYAMY (19)LIRTFSGDVTYNQKFRG (127) SGTVRGFAY (34)  24 DYAMH (117)LIRTYSGGVTYNQKFRG (151) SGTVRGFAY (34)  25 EYAMY (23)LIRTFSGGVSYNQKFRG (138) SGTVRGFAY (34)  26 EYAMH (22)LIRTFSGDVSYNQKFKG (123) SGTVRGFAY (34)  27 DYAMY (21)LIRTYSGGVSYNQKFRG (148) SGTVRGFAY (34)  28 DYAMY (21)VIRTFSGGVTYNQKFRG (32) SGTVRGFAY (34)  29 DYAMY (21)VIRTFSGGVTYNQKFKG (172) SGTVRGFAY (34)  30 DYVMY (35)VIRTFSGGLSYNQKFRG (165) SGTVRGFAY (34)  31 EYAMY (23)LIRTFSGGLTYNQKFKD (133) SGTVRGFAY (34)  32 DYAMY (21)VIRTFSGGVTYNQKFKD (171) SGTVRGFAY (34)  33 GYAMY (19)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34)  34 DYAMY (21)VIRTFSGGVTYNQKFRG (32) SGTVRGFAY (34)  35 GYAMY (19)VIRTFSGDVTYNQKFRG (25) SGTVRGFAY (34)  36 DYAMY (21)VIRTFSGGVSYNQKFRD (168) SGTVRGFAY (34)  37 EYAMY (23)LIRTFSGEVTYNQKFKD (129) SGTVRGFAY (34)  38 GYAMY (19)VIKTYSGGVTYNQKFRG (26) SGTVRGFAY (34)  39 GYAMH (20)LIRTYSGGVSYNQKFRE (27) SGTVRGFAY (34)  40 EYAMY (23)VIRTYSGDLSYNQKFRG (174) SGTVRGFAY (34)  41 DYVMY (35)VIRTFSGGVSYNQKFRG (170) SGTVRGFAY (34)  42 DYAMY (21)VIRTFSGDLTYNQKFQD (28) SGTVRGFAY (34)  43 EYAMY (23)LIRTFSGDVSYNQKFKG (123) SGTVRGFAY (34)  44 EYAMH (22)LIRTYSGDVSYNQKFQG (142) SGTVRGFAY (34)  45 EYAMY (23)LIRTYSGGVSYNQKFQG (147) SGTVRGFAY (34)  46 EYAMY (23)LIRTFSGDLSYNQKFRG (122) SGTVRGFAY (34)  47 DYAMY (21)VIRTYSGGVTYNQKFRD (188) SGTVRGFAD (189)  48 DYAMY (21)LIRTYSGGVTYNQKFKE (149) SGTVRGFAY (34)  49 GYAMY (19)VIRTYSGDVTYNQKFRE (179) SGTVRGFAY (34)  50 DYAMY (21)LIRTFSGGVSYNQKFKE (134) SGTVRGFAY (34)  51 EYAMY (23)VIRTFSGGVTYNQKFKG (172) SGTVRGFAY (34)  52 DYAMY (21)LIRTYSGGVSYNQKFRE (27) SGTVRGFAY (34)  53 EYAMH (22)VIRTYSGGLSYNQKFRG (182) SGTVRGFAY (34)  54 EYAMH (22)LIRTYSGGVSYNQKFQG (147) SGTVRGFAY (34)  55 DYAMY (21)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34)  56 DYAMY (21)VIRTYSGDVSYNQKFRG (177) SGTVRGFAY (34)  57 GYAMY (19)LIRTYSGDVTYNQKFKD (143) SGTVRGFAY (34)  58 DYAMY (21)VIRTYSGGVTYNQKFKG (186) SGTVRGFAY (34)  59 EYAMY (23)LIRTYSGGVSYNQKFRD (31) SGTVRGFAY (34)  60 DYAMY (21)VIKTYSGGVSYNQKFRG (153) SGTVRGFAY (34)  61 EYAMH (22)LIRTYSGGVSYNQKFQE (115) SGTVRGFAY (34)  62 GYVMY (116)VIRTFSGGVSYNQKFQG (167) SGTVRGFAY (34)  63 EYAMY (23)VIRTFSGDVTYNQKFKG (163) SGTVRGFAY (34)  64 DYAMY (21)VIRTYSGDVTYNQKFRG (180) SGTVRGFAY (34)  65 EYAMY (23)VIKTYSGGVTYNQKFRG (26) SGTVRGFAY (34)  66 DYVMY (35)VIRTYSGEVSYNQKFRG (183) SGTVRGFAY (34)  67 EYAMY (23)VIQTFSGDVSYNQKFKG (156) SGTVRGFAY (34)  68 GYAMY (19)LIRTYSGGVTYNQKFRG (151) SGTVRGFAY (34)  69 EYVMH(118)VIRTFSGGVSYNQKFRE (169) SGTVRGFAY (34)  70 GYAMY (19)VIRTYSGDVTYNQKFKD (178) SGTVRGFAY (34)  71 GYAMY (19)VIRTFSGGVTYNQKFRG (32) SGTVRGFAY (34)  72 GYAMY (19)VIRTYSGDVSYNQKFQE (175) SGTVRGFAY (34)  73 GYVMH (119)IIKTYSGGVSYNQKFQG (120) SGTVRGFAY (34)  74 DYAMY (21)VIKTYSGGVTYNQKFKD (154) SGTVRGFAY (34)  75 GYAMY (19)VIRTYSGGVTYNQKFQG (187) SGTVRGFAY (34)  76 DYAMH (117)LIRTFSGDVSYNQKFRE (125) SGTVRGFAY (34)  77 EYAMH (22)LIQTYSGDVSYNQKFRG (121) SGTVRGFAY (34)  78 DYAMY (21)VIKTYSGGVTYNQKFRD (155) SGTVRGFAY (34)  79 EYAMH (22)LIRTYSGGVTYNQKFRE (150) SGTVRGFAY (34)  80 EYAMH (22)LIRTFSGDVSYNQKFRG (126) SGTVRGFAY (34)  81 DYAMY (21)LIRTFSGEVSYNQKFQD (128) SGTVRGFAY (34)  82 GYVMH (119)VIRTFSGGVSYNQKFRG (170) SGTVRGFAY (34)  83 GYAMY (19)VIRTFSGDVSYNQKFRD (161) SGTVRGFAY (34)  84 GYAMY (19)LIRTFSGDVTYNQKFRG (127) SGTVRGFAY (34)  85 EYAMY (23)VIRTYSGGVTYNQKFKD (185) SGTVRGFAY (34)  86 EYAMY (23)VIRTYSGGVTYNQKFRD (188) SGTVRGFAY (34)  87 GYAMY (19)VIRTFSGDLSYNQKFKG (159) SGTVRGFAY (34)  88 EYAMH (22)VIRTYSGDVSYNQKFRG (177) SGTVRGFAY (34)  89 GYAMY (19)VIRTFSGDVTYNQKFRG (25) SGTVRGFAY (34)  90 EYAMY (23)LIRTYSGDLSYNQKFKE (141) SGTVRGFAY (34)  91 EYAMH (22)LIRTYSGGVSYNQKFQE (115) SGTVRGFAY (34)  92 EYAMY (23)LIRTFSGGVTYNQKFQG (139) SGTVRGFAY (34)  93 DYAMH (117)VIQTYSGDVSYNQKFQG (158) SGTVRGFAY (34)  94 GYAMY (19)VIRTFSGGVTYNQKFRD (173) SGTVRGFAY (34)  95 DYAMY (21)LIRTYSGGVSYNQKFRG (148) SGTVRGFAY (34)  96 EYAMY (23)VIRTYSGGLTYNQKFRD (184) SGTVRGFAY (34)  97 EYAMH (22)LIRTFSGGLSYNQKFRD (131) SGTVRGFAY (34)  98 GYAMH (20)VIRTFSGGVSYNQKFQE (166) SGTVRGFAY (34)  99 DYAMH (117)LIRTFSGDLSYNQKFRG (122) SGTVRGFAY (34) 100 EYAMH (22)VIRTFSGGVSYNQKFQG (167) SGTVRGFAY (34) 101 DYAMH (117)LIRTFSGGVSYNQKFQD (136) SGTVRGFAY (34) 102 GYAMY (19)VIRTYSGGVSYNQKFRD (194) SGTVRGFAY (34) 103 GYAMY (19)VIRTYSGDVSYNQKFRG (177) SGTVRGFAY (34) 104 DYAMY (21)LIRTFSGGVSYNQKFRD (137) SGTVRGFAY (34) 105 EYAMY (23)LIRTFSGGVSYNQKFKG (135) SGTVRGFAY (34) 106 DYAMY (21)VIRTFSGDVSYNQKFQE (160) SGTVRGFAY (34) 107 GYAMY (19)VIRTYSGDVSYNQKFRD (176) SGTVRGFAY (34) ²The VH CDRs in Table 2 weredetermined according to Kabat.

TABLE 3 VL FR Amino Acid Sequences³ VL FR1 VL FR2 VL FR3 VL FR4 Antibody(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 231-32-15DIVMTQSPSSLTVTA WYQQKPGQP GVPDRFTGSGSGTDFTLTI FGGGTKLEIK GEKVIMSC (616)PKLLIY (623) SSVQAEDLAVYHC (637) (641) Hum231 #1 DIVMTQSPPTLSLSPWYQQKPGQA GIPARFSGSGSGTDFTLTIS FGQGTKLEIK GERVTLSC (615) PRLLIY (622)SLQPEDFAVYHC (626) (643) Hum231 #2 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (630) (643) pab1964 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643) pab1965 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (639) (643) pab1966 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (631) (643) pab1967 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643) pab1968 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (631) (643) pab1969 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643) pab1970 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (632) (643) pab1971 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (642) (643) pab1972 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643) pab1973 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSDTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (627) (643) pab1975 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (631) (643) pab1976 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (631) (643) pab1977 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (642) (643) pab1979 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (631) (643) pab1980 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (631) (643) pab1981 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (642) (643) pab1983 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (631) (643) pab2159 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSLQAEDVAVYHC (630) (643) pab2160 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643) pab2161 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (639) (643)   1 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYHC (632) (643)   2 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (633) (643)   3 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (638) (643)   4 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (639) (643)   5 DIVMTQSPDSLAAPG WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK ERATINC (610) PKLLIY (618)SSVQAEDVAVYYC (639) (643)   6 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSLQAEDVAVYHC (636) (643)   7 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSLQAEDVAVYHC (630) (643)   8 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643)   9 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSLQAEDVAVYHC (630) (643)  10 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (632) (643)  11 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (632) (643)  12 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSLQAEDVAVYHC (630) (643)  13 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (632) (643)  14 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYYC (633) (643)  15 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  16 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (638) (643)  17 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYHC (632) (643)  18 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYYC (639) (643)  19 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643)  20 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLLY (624)SSVQAEDVAVYHC (620) (643)  21 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643)  22 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYHC (632) (643)  23 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSVQAEDVAVYHC (632) (643)  24 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSVQAEDVAVYHC (632) (643)  25 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSVQAEDVAVYYC (639) (643)  26 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSVQAEDVAVYYC (633) (643)  27 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYHC (630) (643)  28 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYHC (630) (643)  29 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (630) (643)  30 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYYC (631) (643)  31 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  32 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  33 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643)  34 DIVMTQSTDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (617) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  35 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (639) (643)  36 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQEEDVAVYHC (634) (643)  37 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  38 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (631) (643)  39 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643)  40 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643)  41 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYHC (630) (643)  42 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (631) (643)  43 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSVQAEDVAVYHC (638) (643)  44 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYYC (639) (643)  45 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (639) (643)  46 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYHC (636) (643)  47 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643)  48 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  49 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643)  50 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSLQAEDVAVYYC (631) (643)  51 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643)  52 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (630) (643)  53 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643)  54 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643)  55 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (632) (643)  56 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  57 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSLQAEDVAVYHC (630) (643)  58 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643)  59 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643)  60 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (633) (643)  61 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYYC (633) (643)  62 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  63 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYHC (638) (643)  64 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643)  65 DIVMTQSPDSLPVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (612) PKMLIY (619)SFVQAEDVAVYYC (628) (643)  66 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643)  67 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (639) (643)  68 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSLQAEDVAVYYC (642) (643)  69 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYYC (633) (643)  70 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (630) (643)  71 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643)  72 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYHC (630) (643)  73 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYHC (630) (643)  74 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYHC (632) (643)  75 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYYC (642) (643)  76 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSVQAEDVAVYHC (632) (643)  77 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (639) (643)  78 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYHC (638) (643)  79 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSLQAEDVAVYYC (631) (643)  80 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYYC (642) (643)  81 DIVMTQSPDSLSVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (613) PKLLIY (623)SSVQAEDVAVYHC (632) (643)  82 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSLQAEDVAVYYC (631) (643)  83 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (633) (643)  84 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  85 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (633) (643)  86 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (633) (643)  87 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (632) (643)  88 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYYC (633) (643)  89 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (636) (643)  90 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYHC (636) (643)  91 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (632) (643)  92 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTNFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYHC (635) (643)  93 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSLQAEDVAVYHC (636) (643)  94 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYHC (638) (643)  95 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSVQAEDVAVYHC (632) (643)  96 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSLQAEDVAVYHC (630) (643)  97 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (638) (643)  98 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (638) (643)  99 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (624)SSVQAEDVAVYYC (633) (643) 100 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKMLIY (619)SSVQAEDVAVYHC (632) (643) 101 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYHC (632) (643) 102 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYYC (639) (643) 103 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSLQAEDVAVYHC (630) (643) 104 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSFQAEDVAVYHC (629) (643) 105 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (623)SSVQAEDVAVYYC (633) (643) 106 DIVMTQSPDSLAVSL WYQQKPGQPGVPDRFSGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKSLIY (625)SSVQAEDVAVYHC (632) (643) 107 DIVMTQSPDSLAVSL WYHQKPGQPGVPDRFTGSGSGTDFTLTI FGQGTKLEIK GERATINC (611) PKLLIY (618)SSVQAEDVAVYYC (639) (643) ³The VL framework regions described in Table 3are determined based upon the boundaries of the Kabat numbering systemfor CDRs. In other words, the VL CDRs are determined by Kabat and theframework regions are the amino acid residues surrounding the CDRs inthe variable region in the format FR1, CDR1, FR2, CDR2, FR3, CDR3, andFR4.

TABLE 4 VH FR Amino Acid Sequences⁴ VH FR1 VH FR2 VH FR3 VH FR4 Antibody(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 231-32-15QVQLLQSGTELVRPGV WVKQSHAKSL KATMTVDKSSSIAYMEL WGQGTLVTV SVKISCKGSGYTFTEWIG (652) ARLSSEDSAIYYCAK SS (668) (645) (658) Hum231#1 QVQLVQSGAEVKKPGWVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (649) (659) Hum231#2 QVQLVQSGAEVKKPGWVVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (649) (659) pab1964 QVQLVQSGAEVKKPG WVRQAPGQGRVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT MEWIG (655) SRLRSDDTAVYYCAKSS (668) (648) (663) pab1965 QVQLVQSGAEAKKPG WVRQAPGQG RVTMTVDTSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (646)(663) pab1966 QVQLVQSGTEVKKPGA WVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTVSVKVSCKGSGYTFT EWIG (653) SRLRSDDTAVYYCAK SS (668) (651) (662) pab1967QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLITVS ASVKVSCKGSGYTFTEWMG (654) SRLRSDDTAVYYCAK S (667) (649) (670) pab1968 QVQLVQSGTEVKKPGAWVRQAPGQGL RVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (650) (663) pab1969 QVQLVQSGAEVKKPG WVRQAPGQGLRATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (648) (670) pab1970 QVQLVQSGTEVKKPGA WVRQAPGQGLRVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT EWIG (653) SRLRSDDTAVYYCAKSS (668) (650) (663) pab1971 QVQLVQSGTEVKKPGA WVRQAPGQGRVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAKSS (668) (651) (663) pab1972 QVQLVQSGAEVKKPG WVRQAPGQGLRVTMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (648) (662) pab1973 QVQLVQSGAEVKKPG WVRQAPGQG RATMTVDTSISTAYMELWGQGTLVTV ASVKVSCKASGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (648)(670) pab1975 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTVASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (648) (670) pab1976QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFTEWMG (654) SRLRSDDTAVYYCAK SS (668) (648) (670) pab1977 QVQLVQSGAEVKKPGWVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (668) (648) (670) pab1979 QVQLVQSGAEVKKPG WVRQAPGQGLRVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (648) (663) pab1980 QVQLVQSGAEVKKPG WVRQAPGQGLRVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (648) (663) pab1981 QVQLVQSGAEVKKPG WVRQAPGQGLRVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (648) (663) pab1983 QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDTSISTAYMELWGQGTLVTV ASVKVSCKASGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (648)(663) pab2159 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTVASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (648) (659) pab2160QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFTMEWIG (655) SRLRSDDTAVYYCAK SS (668) (651) (663) pab2161 QVQLVQSGAEVKKPGWVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (649) (670)   1 QVQLVQSGTEVKKPGA WVRQAPGQGLRATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFT EWIG (653) SRLRSDDTAVYYCAKSS (668) (651) (670)   2 QVQLVQSGAEVKKPG WVRQAPGQG RATMTVDKSISTAYMELWGQGTLVTV ASVKVSCKASGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (648)(659)   3 QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDKSISTAYMEL WGQGTLVTVASVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (649) (662)   4QVQLVQSGAEVKKPG WVRQAPGQSL RVTMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFTEWMG (657) SRLRSDDTAVYYCAK SS (668) (648) (662)   5 QVQLVQSGAEVKKPGWVRQAPGQGL RVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (668) (649) (663)   6 QVQLVQSGAEVKKPG WVRQAPGQGLRATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (649) (670)   7 QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDTSISTAYMELWGQGTLVTV ASVKVSCKASGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (648)(663)   8 QVQLVQSGAEVKKPG WVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTVASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (649) (662)   9QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFTEWMG (654) SRLRSDDTAVYYCAK SS (668) (648) (659)  10 QVQLVQSGAEVKKPGWVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (668) (648) (662)  11 QVQLVQSGAEVKKPG WVRQAPGQGLRVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (649) (663)  12 QVQLVQSGAEVKKPG WVRQAPGQGL RVTMTVDTSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT EWIG (653) SRLRSDDTAVYYCAK SS (668) (649)(663)  13 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTVASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (648) (659)  14QVQLVQSGTEVKKPGA WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFTEWMG (654) SRLRSDDTAVYYCAK SS (668) (651) (670)  15 QVQLVQSGAEVKKPGWVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (649) (659)  16 QVQLVQSGTEVKKPGA WVRQAPGQGLRVTMTVDKSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT EWIG (653) SRLRSDDTAVYYCAKSS (668) (650) (662)  17 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDKSISTAYMELWGQGTLVTV ASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (648)(659)  18 QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDTSISTAYMEL WGQGTLVTVSVKVSCKASGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (650) (663)  19QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFTEWMG (654) SRLRSDDTAVYYCAK SS (668) (649) (670)  20 QVQLVQSGTEVKKPGAWVRQAPGQGL RVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (650) (663)  21 QVQLVQSGTEVKKPGA WVRQAPGQGRVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAKSS (668) (651) (663)  22 QVQLVQSGAEVKKPG WVRQAPGQG RATMTVDTSISTAYMELWGQGTLVTV ASVKVSCKASGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (648)(670)  23 QVQLVQSGTEVKKPGA WVRQAPGQG RATMTVDKSISTAYMEL WGQGTLVTVSVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (651) (659)  24QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDKSISTAYMEL WGQGTLVTV ASVKASCKGSGYTFTMEWIG (655) SRLRSDDTAVYYCAK SS (668) (647) (662)  25 QVQLVQSGAEVKKPGWVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (668) (648) (670)  26 QVQLVQSGTEVKKPGA WVRQAPGQGRVTMTVDKSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAKSS (668) (651) (662)  27 QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDTSISTAYMELWGQGTLVTV SVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (651)(663)  28 QVQLVQSGTEVKKPGA WVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTVSVKVSCKGSGYTFT EWIG (653) SRLRSDDTAVYYCAK 88 (668) (651) (659)  29QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFTEWIG (653) SRLRSDDTAVYYCAK SS (668) (649) (670)  30 QVQLVQSGAEVKKPGWVRQAPGQGL RVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (668) (649) (663)  31 QVQLVQSGTEVKKPGA WVRQAPGQGLRVTMTVDKSISTAYMEL WGQGIPVTVS SVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAKS (664) (651) (662)  32 QVQLVQSGAEVKKPG WVRQAPGQG RATMTVDKSISTAYMELWGQGTLVTV ASVKVSCKASGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (648)(659)  33 QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDTSISTAYMEL WGQGTLVTVASVKVSCKASGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (648) (663)  34QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFTMEWIG (655) SRLRSDDTAVYYCAK SS (668) (648) (663)  35 QVQLVQSGAEAKKPGWVRQAPGQG RVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT MEWIG (655)SRLRSDDTAVYYCAK SS (668) (646) (663)  36 QVQLVQSGAEVKKPG WVRQAPGQGRVTMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAKSS (668) (649) (662)  37 QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDTSISTAYMELWGQGTLVTV SVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (651)(663)  38 QVQLVQSGTEVKKPGA WVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTVSVKVSCKGSGYTFT EWIG (653) SRLRSDDTAVYYCAK SS (668) (651) (662)  39QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLITVS ASVKVSCKGSGYTFTEWMG (654) SRLRSDDTAVYYCAK S (667) (649) (670)  40 QVQLVQSGAEVKKPGWVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (648) (662)  41 QVQLVQSGAEVKKPG WVRQAPGQGLRATMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (649) (659)  42 QVQLVQSGTEVKKPGA WVRQAPGQGL RVTMTVDTSISTAYMELWGQGTLVTV SVKVSCKASGYTFT EWIG (653) SRLRSDDTAVYYCAK SS (668) (650) (663) 43 QVQLVQSGAEVKKPG WVRQAPGQG RATMTVDKSISTAYMEL WGQGTLVTVASVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (649) (659)  44QVQLVQSGTEVKKPGA WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFTEWMG (654) SRLRSDDTAVYYCAK SS (668) (651) (670)  45 QVQLVQSGAEVKKPGWVRQAPGQGL RVTMTVDTSISTAYMEL WGQGTFVTV ASVKVSCKGSGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (665) (649) (663)  46 QVQLVQSGAEVKKPG WVRQAPGQGLRVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (648) (663)  47 QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDKSISTAYMELWGQGTLVTV ASVKVSCKASGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (648)(662)  48 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTVASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (648) (659)  49QVQLVQSGTEVKKPGA WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFTEWMG (654) SRLRSDDTAVYYCAK SS (668) (651) (670)  50 QVQLVQSGTEVKKPGAWVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (650) (662)  51 QVQLVQSGTEVKKPGA WVRQAPGQGLRATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (650) (670)  52 QVQLVQSGTEVKKPGA WVRQAPGQGL RVTMTVDKSISTAYMELWGQGTLVTV SVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (651) (662) 53 QVQLVQSGTEVKKPGA WVRQAPGQG RATMTVDKSISTAYMEL WGQGTLVTVSVKVSCKASGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (650) (659)  54QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFTEWMG (654) SRLRSDDTAVYYCAK SS (668) (648) (670)  55 QVQLVQSGTEVKKPGAWVRQAPGQGL RVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (650) (663)  56 QVQLVQSGTEVKKPGA WVRQAPGQGRVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAKSS (668) (651) (663)  57 QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDTSISTAYMELWGQGTLVTV SVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (651)(663)  58 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTVASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (649) (659)  59QVQLVQSGAEVKKPG WVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFTEWMG (654) SRLRSDDTAVYYCAK SS (668) (648) (662)  60 QVQLVQSGAEVKKPGWVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (668) (649) (659)  61 QVQLVQSGTEVKKPGA WVRQAPGQGLRVTMTVDKSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (650) (662)  62 QVQLVQSGAEVKKPG WVRQAPGQG RATMTVDKSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (649)(659)  63 QVQLVQSGAEVKKPG WVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTVASVKVSCKASGYTFT EWIG (653) SRLRSDDTAVYYCAK 88 (668) (648) (662)  64QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFTEWMG (654) SRLRSDDTAVYYCAK SS (668) (648) (670)  65 QVQLVQSGAEVKKPGWVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (668) (649) (662)  66 QVQLVQSGTEVKKPGA WVRQAPGQGLRVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (651) (663)  67 QVQLVQSGAEVKKPG WVRQAPGQGL RVTMTVDKSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (649)(662)  68 QVQLVQSGTEVKKPGA WVRQAPGQGL RVTMTVDTSISTAYMEL WGQGTLVTVSVKVSCKGSGYTFT EWIG (653) SRLRSDDTAVYYCAK SS (668) (651) (663)  69QVQLVQSGTEVKKPGA WVRQAPGQG RATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFTMEWMG (656) SRLRSDDTAVYYCAK SS (668) (651) (670)  70 QVQLVQSGTEVKKPGAWVRQAPGQG RATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT MEWIG (655)SRLRSDDTAVYYCAK SS (668) (650) (670)  71 QVQLVQSGAEVKKPG WVRQAPGQGRATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT MEWMG (656) SRLRSDDTAVYYCAKSS (668) (648) (670)  72 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (649)(670)  73 QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDKSISTAYMEL WGQGTLVTVASVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (649) (662)  74QVQLVQSGTEVKKPGA WVRQAPGQG RATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFTMEWIG (655) SRLRSDDTAVYYCAK SS (668) (651) (670)  75 QVQLVQSGTEVKKPGAWVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (651) (662)  76 QVQLVQSGAGVKKPG WVRQAPGQGLRATMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (644) (659)  77 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT EWIG (653) SRLRSDDTAVYYCAK SS (668) (649)(670)  78 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMEL WGRGTLVTVSASVKVSCKGSGYTFT EWIG (653) SRLRGDDTAVYYCAK S (669) (649) (661)  79QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDKSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFTMEWMG (656) SRLRSDDTAVYYCAK SS (668) (651) (662)  80 QVQLVQSGTEVKKPGAWVRQAPGQG RVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT MEWMG (656)SRLRSDDTAVYYCAK SS (668) (650) (663)  81 QVQLVQSGTEVKKPGA WVRQAPGQGLRATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (650) (670)  82 QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDKSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (649)(662)  83 QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDTSISTAYMEL WGQGTLVTVASVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (649) (663)  84QVQLVQSGTEVKKPGA WVRQAPGQG RATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFTMEWMG (656) SRLRSDDTAVYYCAK SS (668) (651) (670)  85 QVQLVQSGAEVKKPGWVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (668) (648) (670)  86 QVQLVQSGAEVKKPG WVRQAPGQGRATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAKSS (668) (649) (670)  87 QVQLVQSGAEVKKPG WVRQAPGQG RATMTVDTSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (649)(670)  88 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDKSISTAYMEL WGQGTLVTVASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (649) (659)  89QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDKSISTAYMEL WGQGTLVTV SVKVSCKASGYTFTMEWMG (656) SRLRSDDTAVYYCAK SS (668) (650) (662)  90 QVQLVQSGAEVKKPGWVRQAPGQGL RATMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWIG (653)SRLRSDDTAVYYCAK SS (668) (648) (670)  91 QVQLVQSGTEVKKPGA WVRQAPGQGRATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAKSS (668) (651) (670)  92 QVQLVQSGAEVKKPG WVRQAPGQGL RATMTVDTSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT EWMG (654) SRLQSDDTAVYYCAK SS (668) (649)(660)  93 QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDKSISTAYMEL WGQGTLVTVSVKVSCKASGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (650) (662)  94QVQLVQSGTEVKKPGA WVRQAPGQGL RVTMTVDKSISTAYMEL WGQGTFVTV SVKVSCKGSGYTFTEWIG (653) SRLRSDDTAVYYCAK SS (666) (651) (662)  95 QVQLVQSGAEVKKPGWVRQAPGQGL RVTMTVDTSISTAYMEL WGQGTFVTV ASVKVSCKGSGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (666) (649) (663)  96 QVQLVQSGAEVKKPG WVRQAPGQGRVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAKSS (668) (649) (663)  97 QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDKSISTAYMELWGQGTLVTV SVKVSCKGSGYTFT MEWIG (655) SRLRSDDTAVYYCAK SS (668) (651)(662)  98 QVQLVQSGAEVKKPG WVRQAPGQG RATMTVDTSISTAYMEL WGQGTLVTVASVKVSCKGSGYTFT MEWMG (656) SRLRSDDTAVYYCAK 88 (668) (649) (670)  99QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFTMEWMG (656) SRLRSDDTAVYYCAK SS (668) (648) (662) 100 QVQLVQSGTEVKKPGAWVRQAPGQG RATMTVDTSISTAYMEL WGQGTLVTV SVKVSCKASGYTFT MEWMG (656)SRLRSDDTAVYYCAK SS (668) (650) (670) 101 QVQLVQSGAEVKKPG WVRQAPGQGLRVTMTVDKSISTAYMEL WGQGTLVTV ASVKVSCKASGYTFT EWMG (654) SRLRSDDTAVYYCAKSS (668) (648) (662) 102 QVQLVQSGTEVKKPGA WVRQAPGQGL RVTMTVDTSISTAYMELWGQGTLVTV SVKVSCKASGYTFT EWIG (653) SRLRSDDTAVYYCAK SS (668) (650) (663)103 QVQLVQSGAEVKKPG WVRQAPGQG RVTMTVDTSISTAYMEL WGQGTLVTVASVKVSCKASGYTFT MEWMG (656) SRLRSDDTAVYYCAK SS (668) (648) (663) 104QVQLVQSGTEVKKPGA WVRQAPGQG RVTMTVDTSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFTMEWMG (656) SRLRSDDTAVYYCAK SS (668) (651) (663) 105 QVQLVQSGAEVKKPGWVRQAPGQGL RVTMTVDTSISTAYMEL WGQGTLVTV ASVKVSCKGSGYTFT EWMG (654)SRLRSDDTAVYYCAK SS (668) (649) (663) 106 QVQLVQSGTEVKKPGA WVRQAPGQGLRATMTVDKSISTAYMEL WGQGTLVTV SVKVSCKGSGYTFT EWIG (653) SRLRSDDTAVYYCAKSS (668) (651) (659) 107 QVQLVQSGAEVKKPG WVRQAPGQGL RVTMTVDTSISTAYMELWGQGTLVTV ASVKVSCKGSGYTFT EWMG (654) SRLRSDDTAVYYCAK SS (668) (649)(663) ⁴The VH framework regions described in Table 4 are determinedbased upon the boundaries of the Kabat numbering system for CDRs. Inother words, the VH CDRs are determined by Kabat and the frameworkregions are the amino acid residues surrounding the CDRs in the variableregion in the format FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprises alight chain variable region (VL) comprising:

(a) a VL CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence KSSQSX₁X₂X₃X₄X₅X₆X₇KX₈YLX₉ (SEQ ID NO: 4),wherein:

X₁ is L, A, V, I, P, F or M

X₂ is L, A, V, I, P, F, M or S

X₃ is N, G, Q, S, T, C, W, Y or A

X₄ is S, G, N, Q, T, C, W, Y or A

X₅ is G, N, Q, S, T, C, W, Y or A

X₆ is N, G, Q, S, T, C, W, Y or A

X₇ is Q, G, N, S, T, C, W, Y or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is T, G, N, Q, S, C, W, Y, V, I or A; and/or

(b) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁ASTRX₂X₃(SEQ ID NO: 5), wherein:

X₁ is W, G, N, Q, S, T, C, Y, F, H or A

X₂ is E, D or A

X₃ is S, G, N, Q, T, C, W, Y or A; and/or

(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence QX₁X₂YX₃X₄PYT (SEQ ID NO: 6), wherein:

X₁ is N, G, Q, S, T, C, W or Y

X₂ is D, E or Y

X₃ is S, G, N, Q, T, C, W, Y or A

X₄ is Y, G, N, Q, S, T, C, W, F, H, L, or A.

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two, or all three of the VL CDRs above. Incertain embodiments, the antibody or antigen-binding fragment thereofcomprises the VL CDR1 of one of the antibodies in Table 5. In someembodiments, the antibody or antigen-binding fragment thereof comprisesthe VL CDR2 of one of the antibodies in Table 5. In certain embodiments,the antibody or antigen-binding fragment thereof comprises the VL CDR3of one of the antibodies in Table 5. In certain embodiments, theantibody or antigen-binding fragment thereof comprises one, two or allthree of the VL CDRs of one of the antibodies in Table 5 (e.g., the VLCDRs in one row of Table 5, for example, all of the VL CDRs are fromantibody 231-32-15). In some embodiments, the antibody orantigen-binding fragment thereof comprises the VL framework regionsdescribed herein. In specific embodiments, the antibody orantigen-binding fragment thereof comprises the VL framework regions(FRs) of an antibody set forth in Table 7 (e.g., one, two, three, orfour of the framework regions in one row of Table 7).

In another embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR), comprises a heavy chain variable region (VH)comprising:

(a) a VH CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁YX₂MX₃ (SEQ ID NO: 1), wherein

X₁ is D, E, G or A

X₂ is A, V, L, I, P, F, M or Y

X₃ is Y, G, N, Q, S, T, C, W, F or H; and/or

(b) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁IX₂X₃X₄SGX₅X₆X₇YX₈QKFX₉X₁₀ (SEQ ID NO: 2),wherein

X₁ is V, A, L, I, P, F, M or T

X₂ is R, K, H, Q or A

X₃ is T, G, N, Q, S, C, W, Y, V, I or P

X₄ is Y, G, N, Q, S, T, C, W, F, H, or A

X₅ is D, E, G or A

X₆ is V, A, L, I, P, F, M or T

X₇ is T, G, N, Q, S, C, W, Y, V, I, P or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is K, R, H, Q or A

X₁₀ is D, E, G or A; and/or

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence SGTVRGX₁X₂X₃ (SEQ ID NO: 3), wherein

X₁ is F, A, V, L, I, P, M, Y, W, H or S

X₂ is A, or D

X₃ is Y, G, N, Q, S, T, C, W, F, H or V.

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two or all three of the VH CDRs above. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesthe VH CDR1 of one of the antibodies in Table 6. In some embodiments,the antibody or antigen-binding fragment thereof comprises the VH CDR2of one of the antibodies in Table 6. In certain embodiments, theantibody or antigen-binding fragment thereof comprises the VH CDR3 ofone of the antibodies in Table 6. In some embodiments, the antibody orantigen-binding fragment thereof comprises one, two or all three of VHCDRs of one of the antibodies in Table 6 (e.g., the VH CDRs in one rowof Table 6, for example, all of the VH CDRs are from the antibody231-32-15). In some embodiments, the antibody or antigen-bindingfragment thereof comprises the VH frameworks described herein. Inspecific embodiments, the antibody or antigen-binding fragment thereofcomprises the VH framework regions of an antibody set forth in Table 8(e.g., one, two, three or four of the framework regions in one row ofTable 8).

TABLE 5 VL CDR Amino Acid Sequences¹ VL CDR2 VL CDR3 AntibodyVL CDR1 (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 231-32-15KSSQSLLNSGNQKNYLT (16) WASTRES (17) QNDYSYPYT (18) Hum231#1KSSQSLLNSGNQKNYLT (16) WASTRES (17) QNDYSYPYT (18) Hum231#2KSSQSLLNSGNQKNYLT (16) WASTRES (17) QNDYSYPYT (18) pab1964KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSYPYT (106) pab1965KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab1966KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab1967KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNEYSFPYT (108) pab1968KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) pab1969KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109) pab1970KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109) pab1971KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107) pab1972KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107) pab1973KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107) pab1975KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab1976KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) pab1977KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107) pab1979KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab1980KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) pab1981KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107) pab1983KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab2159KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109) pab2160KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107) pab2161KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)   1KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)   2KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSFPYT (108)   4KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)   5KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNEYSFPYT (108)   6KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)   9KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  10KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  11KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSFPYT (109)  15KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  16KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  18KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  20KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  21KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  25KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  29KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  31KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  33KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSYPYT (106)  34KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  35KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  36KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  37KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSYPYT (106)  38KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  39KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNEYSFPYT (108)  42KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  43KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  45KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  46KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  47KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  49KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  50KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  52KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  54KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  55KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  58KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  59KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  61KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  68KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  70KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  71KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSYPYT (107)  75KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107)  76KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSFPYT (109)  78KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  79KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  80KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109)  85KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  86KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  91KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNEYSFPYT (108)  92KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  94KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSYPYT (107)  95KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNDYSFPYT (109)  96KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107)  97KSSQSLLNSGNQKNYLT (102) WASTRES (105) QNEYSFPYT (108) 101KSSQSLLNSSNQKNYLT (103) WASTRES (105) QNDYSFPYT (109) 102KSSQSLLNSSNQKNYLS (104) WASTRES (105) QNDYSYPYT (107) 105KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) 107KSSQSLLNSGNQKNYLS (101) WASTRES (105) QNDYSYPYT (107) ¹The VL CDRs inTable 5 are determined according to Kabat.

TABLE 6 VH CDR Amino Acid Sequences² VH CDR1 VH CDR3 Antibody(SEQ ID NO:) VH CDR2 (SEQ ID NO:) (SEQ ID NO:) 231-32-15 DYAMY (13)VIRTYSGDVTYNQKFKD (14) SGTVRGFAY (15) Hum231#l DYAMY (13)VIRTYSGDVTYNQKFKD (14) SGTVRGFAY (15) Hum231#2 DYAMY (13)VIRTYSGDVTYNQKFKD (14) SGTVRGFAY (15) pabl964 GYAMY (19)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34) pabl965 GYAMY (19)VIRTFSGDVTYNQKFRG (25) SGTVRGFAY (34) pabl966 GYAMY (19)VIKTYSGGVTYNQKFRG (26) SGTVRGFAY (34) pabl967 GYAMH (20)LIRTYSGGVSYNQKFRE (27) SGTVRGFAY (34) pabl968 DYAMY (21)VIRTFSGDLTYNQKFQD (28) SGTVRGFAY (34) pabl969 EYAMH (22)LIRTYSGGVSYNQKFQG (29) SGTVRGFAY (34) pabl970 DYAMY (21)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34) pabl971 DYAMY (21)VIRTYSGDVSYNQKFRG (177) SGTVRGFAY (34) pabl972 EYAMY (23)LIRTYSGGVSYNQKFRD (31) SGTVRGFAY (34) pabl973 GYAMY (19)VIRTFSGGVTYNQKFRG (32) SGTVRGFAY (34) pabl975 EYAMH (22)LIRTYSGGVSYNQKFQG (29) SGTVRGFAY (34) pabl976 EYAMH (22)LIRTYSGGVSYNQKFQG (29) SGTVRGFAY (34) pabl977 EYAMH (22)LIRTYSGGVSYNQKFQG (29) SGTVRGFAY (34) pabl979 EYAMH (22)VIRTYSGGVSYNQKFQE (33) SGTVRGFAY (34) pabl980 EYAMH (22)VIRTYSGGVSYNQKFQE (33) SGTVRGFAY (34) pabl981 EYAMH (22)VIRTYSGGVSYNQKFQE (33) SGTVRGFAY (34) pabl983 GYAMY (19)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34) pab2159 GYAMY (19)LIRTYSGEVSYNQKFRG (144) SGTVRGFAY (34) pab2160 GYVMH (119)VIRTFSGDVSYNQKFRE (162) SGTVRGFAY (34) pab2161 EYAMH (22)LIQTYSGDVSYNQKFRG (121) SGTVRGFAY (34)   1 EYAMY (23)VIRTYSGGVTYNQKFQG (187) SGTVRGFAY (34)   2 EYAMH (22)LIRTYSGGVSYNQKFRG (148) SGTVRGFAY (34)   4 EYAMY (23)LIRTFSGDVSYNQKFQD (124) SGTVRGFAY (34)   5 EYAMH (22)LIRTYSGGVTYNQKFRG (151) SGTVRGFAY (34)   6 EYAMY (23)LIRTFSGGVSYNQKFKG (135) SGTVRGFAY (34)   9 GYAMY (19)LIRTYSGEVSYNQKFRG (144) SGTVRGFAY (34)  10 EYAMY (23)LIRTYSGGVSYNQKFRG (148) SGTVRGFAY (34)  11 DYAMH (117)LIRTYSGGVSYNQKFRG (148) SGTVRGFAY (34)  15 DYAMY (21)VIRTFSGDVSYNQKFRE (162) SGTVRGFAY (34)  16 GYAMY (19)LIRTFSGGVTYNQKFRE (140) SGTVRGFAY (34)  18 GYAMY (19)LIRTFSGEVTYNQKFRG (130) SGTVRGFAY (34)  20 DYAMY (21)VIRTFSGDLSYNQKFRG (114) SGTVRGFAY (34)  21 GYVMH (119)VIRTFSGDVSYNQKFRE (162) SGTVRGFAY (34)  25 EYAMY (23)LIRTFSGGVSYNQKFRG (138) SGTVRGFAY (34)  29 DYAMY (21)VIRTFSGGVTYNQKFKG (172) SGTVRGFAY (34)  31 EYAMY (23)LIRTFSGGLTYNQKFKD (133) SGTVRGFAY (34)  33 GYAMY (19)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34)  34 DYAMY (21)VIRTFSGGVTYNQKFRG (32) SGTVRGFAY (34)  35 GYAMY (19)VIRTFSGDVTYNQKFRG (25) SGTVRGFAY (34)  36 DYAMY (21)VIRTFSGGVSYNQKFRD (168) SGTVRGFAY (34)  37 EYAMY (23)LIRTFSGEVTYNQKFKD (129) SGTVRGFAY (34)  38 GYAMY (19)VIKTYSGGVTYNQKFRG (26) SGTVRGFAY (34)  39 GYAMH (20)LIRTYSGGVSYNQKFRE (27) SGTVRGFAY (34)  42 DYAMY (21)VIRTFSGDLTYNQKFQD (28) SGTVRGFAY (34)  43 EYAMY (23)LIRTFSGDVSYNQKFKG (123) SGTVRGFAY (34)  45 EYAMY (23)LIRTYSGGVSYNQKFQG (147) SGTVRGFAY (34)  46 EYAMY (23)LIRTFSGDLSYNQKFRG (122) SGTVRGFAY (34)  47 DYAMY (21)VIRTYSGGVTYNQKFRD (188) SGTVRGFAD (189)  49 GYAMY (19)VIRTYSGDVTYNQKFRE (179) SGTVRGFAY (34)  50 DYAMY (21)LIRTFSGGVSYNQKFKE (134) SGTVRGFAY (34)  52 DYAMY (21)LIRTYSGGVSYNQKFRE (27) SGTVRGFAY (34)  54 EYAMH (22)LIRTYSGGVSYNQKFQG (147) SGTVRGFAY (34)  55 DYAMY (21)LIRTYSGGVTYNQKFQG (24) SGTVRGFAY (34)  58 DYAMY (21)VIRTYSGGVTYNQKFKG (186) SGTVRGFAY (34)  59 EYAMY (23)LIRTYSGGVSYNQKFRD (31) SGTVRGFAY (34)  61 EYAMH (22)LIRTYSGGVSYNQKFQE (115) SGTVRGFAY (34)  68 GYAMY (19)LIRTYSGGVTYNQKFRG (151) SGTVRGFAY (34)  70 GYAMY (19)VIRTYSGDVTYNQKFKD (178) SGTVRGFAY (34)  71 GYAMY (19)VIRTFSGGVTYNQKFRG (32) SGTVRGFAY (34)  75 GYAMY (19)VIRTYSGGVTYNQKFQG (187) SGTVRGFAY (34)  76 DYAMH (117)LIRTFSGDVSYNQKFRE (125) SGTVRGFAY (34)  78 DYAMY (21)VIKTYSGGVTYNQKFRD (155) SGTVRGFAY (34)  79 EYAMH (22)LIRTYSGGVTYNQKFRE (150) SGTVRGFAY (34)  80 EYAMH (22)LIRTFSGDVSYNQKFRG (126) SGTVRGFAY (34)  85 EYAMY (23)VIRTYSGGVTYNQKFKD (185) SGTVRGFAY (34)  86 EYAMY (23)VIRTYSGGVTYNQKFRD (188) SGTVRGFAY (34)  91 EYAMH (22)LIRTYSGGVSYNQKFQE (115) SGTVRGFAY (34)  92 EYAMY (23)LIRTFSGGVTYNQKFQG (139) SGTVRGFAY (34)  94 GYAMY (19)VIRTFSGGVTYNQKFRD (173) SGTVRGFAY (34)  95 DYAMY (21)LIRTYSGGVSYNQKFRG (148) SGTVRGFAY (34)  96 EYAMY (23)VIRTYSGGLTYNQKFRD (184) SGTVRGFAY (34)  97 EYAMH (22)LIRTFSGGLSYNQKFRD (131) SGTVRGFAY (34) 101 DYAMH (117)LIRTFSGGVSYNQKFQD (136) SGTVRGFAY (34) 102 GYAMY (19)VIRTYSGGVSYNQKFRD (194) SGTVRGFAY (34) 105 EYAMY (23)LIRTFSGGVSYNQKFKG (135) SGTVRGFAY (34) 107 GYAMY (19)VIRTYSGDVSYNQKFRD (176) SGTVRGFAY (34) ²The VH CDRs in Table 6 weredetermined according to Kabat.

TABLE 7 VL FR Amino Acid Sequences³ VL FR1 VL FR2 VL FR3 VL FR4 Antibody(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 231-32-DIVMTQSPSSLTVTAG WYQQKPGQPPK GVPDRFTGSGSGTDFTLTISS FGGGTKLEIK 15EKVIMSC (616) LLIY (623) VQAEDLAVYHC (637) (641) Hum231 DIVMTQSPPTLSLSPGWYQQKPGQAPR GIPARFSGSGSGTDFTLTISS FGQGTKLEIK #1 ERVTLSC (615) LLIY (622)LQPEDFAVYHC (626) (643) Hum231 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK #2 ERATINC (611) LLIY (623)LQAEDVAVYHC (630) (643) pab1964 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (618)VQAEDVAVYHC (632) (643) pab1965 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (618)VQAEDVAVYYC (639) (643) pab1966 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (623)LQAEDVAVYYC (631) (643) pab1967 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (623)VQAEDVAVYHC (638) (643) pab1968 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (623)LQAEDVAVYYC (631) (643) pab1969 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (638) (643) pab1970 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (632) (643) pab1971 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (642) (643) pab1972 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYHC (632) (643) pab1973 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSDTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (627) (643) pab1975 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (631) (643) pab1976 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (631) (643) pab1977 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (642) (643) pab1979 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (631) (643) pab1980 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (631) (643) pab1981 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTSS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (642) (643) pab1983 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (631) (643) pab2159 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (624)LQAEDVAVYHC (630) (643) pab2160 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYHC (632) (643) pab2161 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYYC (639) (643)   1 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (619)VQAEDVAVYHC (632) (643)   2 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYYC (633) (643)   4 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYYC (639) (643)   5 DIVMTQSPDSLAAPGE WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  RATINC (610) LLIY (618)VQAEDVAVYYC (639) (643)   6 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (624)LQAEDVAVYHC (636) (643)   9 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (624)LQAEDVAVYHC (630) (643)  10 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (632) (643)  11 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (632) (643)  15 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYHC (636) (643)  16 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYHC (638) (643)  18 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (619)VQAEDVAVYYC (639) (643)  20 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLLY (624)VQAEDVAVYHC (620) (643)  21 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYHC (632) (643)  25 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (624)VQAEDVAVYYC (639) (643)  29 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYHC (630) (643)  31 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYHC (636) (643)  33 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (618)VQAEDVAVYHC (632) (643)  34 DIVMTQSTDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYHC (636) (643)  35 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYYC (639) (643)  36 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (619)VQEEDVAVYHC (634) (643)  37 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYHC (636) (643)  38 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (631) (643)  39 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (638) (643)  42 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (631) (643)  43 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (624)VQAEDVAVYHC (638) (643)  45 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYYC (639) (643)  46 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)LQAEDVAVYHC (636) (643)  47 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (638) (643)  49 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYHC (632) (643)  50 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (624)LQAEDVAVYYC (631) (643)  52 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYHC (630) (643)  54 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (638) (643)  55 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (632) (643)  58 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYHC (632) (643)  59 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYHC (632) (643)  61 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYYC (633) (643)  68 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (624)LQAEDVAVYYC (642) (643)  70 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYHC (630) (643)  71 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (638) (643)  75 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)LQAEDVAVYYC (642) (643)  76 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (624)VQAEDVAVYHC (632) (643)  78 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (619)VQAEDVAVYHC (638) (643)  79 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (619)LQAEDVAVYYC (631) (643)  80 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)LQAEDVAVYYC (642) (643)  85 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYYC (633) (643)  86 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)VQAEDVAVYYC (633) (643)  91 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (632) (643)  92 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTNFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (619)VQAEDVAVYHC (635) (643)  94 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (623)VQAEDVAVYHC (638) (643)  95 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) MLIY (624)VQAEDVAVYHC (632) (643)  96 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK  ERATINC (611) LLIY (618)LQAEDVAVYHC (630) (643)  97 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (618)VQAEDVAVYHC (638) (643) 101 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (618)VQAEDVAVYHC (632) (643) 102 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (623)VQAEDVAVYYC (639) (643) 105 DIVMTQSPDSLAVSLG WYQQKPGQPPKGVPDRFSGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (623)VQAEDVAVYYC (633) (643) 107 DIVMTQSPDSLAVSLG WYHQKPGQPPKGVPDRFTGSGSGTDFTLTISS FGQGTKLEIK ERATINC (611) LLIY (618)VQAEDVAVYYC (639) (643) ³The VL framework regions described in Table 7are determined based upon the boundaries of the Kabat numbering systemfor CDRs. In other words, the VL CDRs are determined by Kabat and theframework regions are the amino acid residues surrounding the CDRs inthe variable region in the format FR1, CDR1, FR2, CDR2, FR3, CDR3, andFR4.

TABLE 8 VH FR Amino Acid Sequences⁴ VH FR1 VH FR2 VH FR3 VH FR4 Antibody(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 231-32-15QVQLLQSGTELVRPGVSVKI WVKQSHAKSLEWI KATMTVDKSSSIAYMELARLS WGQGTLVTVSSSCKGSGYTFT (645) G (652) SEDSAIYYCAK (658) (668) Hum231#1QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWI RATMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (653) SDDTAVYYCAK (659) (668) Hum231#2QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWI RATMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (653) SDDTAVYYCAK (659) (668) pab1964QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (655) SDDTAVYYCAK (663) (668) pab1965QVQLVQSGAEAKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (646) G (655) SDDTAVYYCAK (663) (668) pab1966QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (653) SDDTAVYYCAK (662) (668) pab1967QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLITVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (670) (667) pab1968QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (653) SDDTAVYYCAK (663) (668) pab1969QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (670) (668) pab1970QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (653) SDDTAVYYCAK (663) (668) pab1971QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (656) SDDTAVYYCAK (663) (668) pab1972QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (662) (668) pab1973QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (656) SDDTAVYYCAK (670) (668) pab1975QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (670) (668) pab1976QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (670) (668) pab1977QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (670) (668) pab1979QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (663) (668) pab1980QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (663) (668) pab1981QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (663) (668) pab1983QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (655) SDDTAVYYCAK (663) (668) pab2159QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (659) (668) pab2160QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (655) SDDTAVYYCAK (663) (668) pab2161QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWI RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (653) SDDTAVYYCAK (670) (668)   1QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (653) SDDTAVYYCAK (670) (668)   2QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWI RATMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (655) SDDTAVYYCAK (659) (668)   4QVQLVQSGAEVKKPGASVKV WVRQAPGQSLEWM RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (657) SDDTAVYYCAK (662) (668)   5QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (663) (668)   6QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (670) (668)   9QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (659) (668)  10QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (662) (668)  11QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (663) (668)  15QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWI RATMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (653) SDDTAVYYCAK (659) (668)  16QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (653) SDDTAVYYCAK (662) (668)  18QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (655) SDDTAVYYCAK (663) (668)  20QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (653) SDDTAVYYCAK (663) (668)  21QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (655) SDDTAVYYCAK (663) (668)  25QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (670) (668)  29QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWI RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (653) SDDTAVYYCAK (670) (668)  31QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDKSISTAYMELSRLR WGQGIPVTVSS  SCKGSGYTFT (651) G (654) SDDTAVYYCAK (662) (664)  33QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (655) SDDTAVYYCAK (663) (668)  34QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (655) SDDTAVYYCAK (663) (668)  35QVQLVQSGAEAKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (646) G (655) SDDTAVYYCAK (663) (668)  36QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (655) SDDTAVYYCAK (662) (668)  37QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (655) SDDTAVYYCAK (663) (668)  38QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (653) SDDTAVYYCAK (662) (668)  39QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLITVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (670) (667)  42QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (653) SDDTAVYYCAK (663) (668)  43QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWI RATMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (655) SDDTAVYYCAK (659) (668)  45QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDTSISTAYMELSRLR WGQGTFVTVSSSCKGSGYTFT (649) G (653) SDDTAVYYCAK (663) (665)  46QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (663) (668)  47QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWM RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (656) SDDTAVYYCAK (662) (668)  49QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (654) SDDTAVYYCAK (670) (668)  50QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (653) SDDTAVYYCAK (662) (668)  52QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (654) SDDTAVYYCAK (662) (668)  54QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (670) (668)  55QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (653) SDDTAVYYCAK (663) (668)  58QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (659) (668)  59QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (662) (668)  61QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (654) SDDTAVYYCAK (662) (668)  68QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (653) SDDTAVYYCAK (663) (668)  70QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWI RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (655) SDDTAVYYCAK (670) (668)  71QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (656) SDDTAVYYCAK (670) (668)  75QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (653) SDDTAVYYCAK (662) (668)  76QVQLVQSGAGVKKPGASVKV WVRQAPGQGLEWM RATMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (644) G (654) SDDTAVYYCAK (659) (668)  78QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWI RATMTVDTSISTAYMELSRLR WGRGTLVTVSSSCKGSGYTFT (649) G (653) GDDTAVYYCAK (661) (669)  79QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWM RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (656) SDDTAVYYCAK (662) (668)  80QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (656) SDDTAVYYCAK (663) (668)  85QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (670) (668)  86QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (656) SDDTAVYYCAK (670) (668)  91QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWM RATMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (656) SDDTAVYYCAK (670) (668)  92QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RATMTVDTSISTAYMELSRLQ WGQGTLVTVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (660) (668)  94QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDKSISTAYMELSRLR WGQGTFVTVSSSCKGSGYTFT (651) G (653) SDDTAVYYCAK (662) (666)  95QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDTSISTAYMELSRLR WGQGTFVTVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (663) (666)  96QVQLVQSGAEVKKPGASVKV WVRQAPGQGMEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (656) SDDTAVYYCAK (663) (668)  97QVQLVQSGTEVKKPGASVKV WVRQAPGQGMEWI RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (651) G (655) SDDTAVYYCAK (662) (668) 101QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDKSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (648) G (654) SDDTAVYYCAK (662) (668) 102QVQLVQSGTEVKKPGASVKV WVRQAPGQGLEWI RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKASGYTFT (650) G (653) SDDTAVYYCAK (663) (668) 105QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (663) (668) 107QVQLVQSGAEVKKPGASVKV WVRQAPGQGLEWM RVTMTVDTSISTAYMELSRLR WGQGTLVTVSSSCKGSGYTFT (649) G (654) SDDTAVYYCAK (663) (668) ⁴The VH frameworkregions described in Table 8 are determined based upon the boundariesthe Kabat numbering system for CDRs. In other words, the VH CDRs aredetermined by and the framework regions are the amino acid residuessurrounding the CDRs in the variable region in the format FR1, CDR1,FR2, CDR2, FR3, CDR3, and FR4.

In another embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR), comprises:

(a) a VL CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence KSSQSX₁X₂X₃X₄X₅X₆X₇KX₈YLX₉ (SEQ ID NO: 4),wherein:

X₁ is L, A, V, I, P, F or M

X₂ is L, A, V, I, P, F, M or S

X₃ is N, G, Q, S, T, C, W, Y or A

X₄ is S, G, N, Q, T, C, W, Y or A

X₅ is G, N, Q, S, T, C, W, Y or A

X₆ is N, G, Q, S, T, C, W, Y or A

X₇ is Q, G, N, S, T, C, W, Y or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is T, G, N, Q, S, C, W, Y, V, I or A; and/or

(b) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁ASTRX₂X₃(SEQ ID NO: 5), wherein:

X₁ is W, G, N, Q, S, T, C, Y, F, H or A

X₂ is E, D or A

X₃ is S, G, N, Q, T, C, W, Y or A; and/or

(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence QX₁X₂YX₃X₄PYT (SEQ ID NO: 6), wherein:

X₁ is N, G, Q, S, T, C, W or Y

X₂ is D, E or Y

X₃ is S, G, N, Q, T, C, W, Y or A

X₄ is Y, G, N, Q, S, T, C, W, F, H, L, or A; and/or

(d) a VH CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁YX₂MX₃ (SEQ ID NO: 1), wherein

X₁ is D, E, G or A

X₂ is A, V, L, I, P, F, M or Y

X₃ is Y, G, N, Q, S, T, C, W, F or H; and/or

(e) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁IX₂X₃X₄SGX₅X₆X₇YX₈QKFX₉X₁₀ (SEQ ID NO: 2),wherein

X₁ is V, A, L, I, P, F, M or T

X₂ is R, K, H, Q or A

X₃ is T, G, N, Q, S, C, W, Y, V, I or P

X₄ is Y, G, N, Q, S, T, C, W, F, H, or A

X₅ is D, E, G or A

X₆ is V, A, L, I, P, F, M or T

X₇ is T, G, N, Q, S, C, W, Y, V, I, P or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is K, R, H, Q or A

X₁₀ is D, E, G or A; and/or

(f) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence SGTVRGX₁X₂X₃ (SEQ ID NO: 3), wherein

X₁ is F, A, V, L, I, P, M, Y, W, H or S

X₂ is A, or D

X₃ is Y, G, N, Q, S, T, C, W, F, H or V.

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two, three, four, five or all six of the CDRsabove. In certain embodiments, the antibody or antigen-binding fragmentthereof comprises the VL CDR1 of one of the antibodies in Table 1. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises the VL CDR2 of one of the antibodies in Table 1. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesthe VL CDR3 of one of the antibodies in Table 1. In some embodiments,the antibody or antigen-binding fragment thereof comprises the VH CDR1of one of the antibodies in Table 2. In some embodiments, the antibodyor antigen-binding fragment thereof comprises the VH CDR2 of one of theantibodies in Table 2. In certain embodiments, the antibody orantigen-binding fragment thereof comprises the VH CDR3 of one of theantibodies in Table 2. In some embodiments, the antibody orantigen-binding fragment thereof comprises one, two or all three of theVH CDRs of one of the antibodies in Table 2 (e.g., the VH CDRs in onerow of Table 2, for example, all of the VH CDRs are from the antibody231-32-15). In certain embodiments, the antibody or antigen-bindingfragment thereof comprises one, two or all three of the VL CDRs of oneof the antibodies in Table 1 (e.g., the VL CDRs in one row of Table 1,for example, all of the VLCDRs are from the antibody 231-32-15).

In another embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR), comprises a light chain variable region (VL) and aheavy chain variable region (VH), wherein

(i) the VL comprises:

(a) a VL CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence KSSQSX₁X₂X₃X₄X₅X₆X₇KX₈YLX₉ (SEQ ID NO: 4),wherein:

X₁ is L, A, V, I, P, F or M

X₂ is L, A, V, I, P, F, M or S

X₃ is N, G, Q, S, T, C, W, Y or A

X₄ is S, G, N, Q, T, C, W, Y or A

X₅ is G, N, Q, S, T, C, W, Y or A

X₆ is N, G, Q, S, T, C, W, Y or A

X₇ is Q, G, N, S, T, C, W, Y or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is T, G, N, Q, S, C, W, Y, V, I or A; and/or

(b) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁ASTRX₂X₃(SEQ ID NO: 5), wherein:

X₁ is W, G, N, Q, S, T, C, Y, F, H or A

X₂ is E, D or A

X₃ is S, G, N, Q, T, C, W, Y or A; and/or

(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence QX₁X₂YX₃X₄PYT (SEQ ID NO: 6), wherein:

X₁ is N, G, Q, S, T, C, W or Y

X₂ is D, E or Y

X₃ is S, G, N, Q, T, C, W, Y or A

X₄ is Y, G, N, Q, S, T, C, W, F, H, L, or A; and

(ii) the VH comprises:

(a) a VH CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁YX₂MX₃ (SEQ ID NO: 1), wherein

X₁ is D, E, G or A

X₂ is A, V, L, I, P, F, M or Y

X₃ is Y, G, N, Q, S, T, C, W, F or H; and/or

(b) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁IX₂X₃X₄SGX₅X₆X₇YX₈QKFX₉X₁₀ (SEQ ID NO: 2),wherein

X₁ is V, A, L, I, P, F, M or T

X₂ is R, K, H, Q or A

X₃ is T, G, N, Q, S, C, W, Y, V, I or P

X₄ is Y, G, N, Q, S, T, C, W, F, H, or A

X₅ is D, E, G or A

X₆ is V, A, L, I, P, F, M or T

X₇ is T, G, N, Q, S, C, W, Y, V, I, P or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is K, R, H, Q or A

X₁₀ is D, E, G or A; and/or

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence SGTVRGX₁X₂X₃ (SEQ ID NO: 3), wherein

X₁ is F, A, V, L, I, P, M, Y, W, H or S

X₂ is A, or D

X₃ is Y, G, N, Q, S, T, C, W, F, H or V.

In specific embodiments, the VL comprises two or all three of the VLCDRs above and/or the VH comprises two or all three of the VH CDRsabove. In certain embodiments, the antibody or antigen-binding fragmentthereof comprises the VL CDR1 of one of the antibodies in Table 1. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises the VL CDR2 of one of the antibodies in Table 1. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesthe VL CDR3 of one of the antibodies in Table 1. In some embodiments,the antibody or antigen-binding fragment thereof comprises the VH CDR1of one of the antibodies in Table 2. In some embodiments, the antibodyor antigen-binding fragment thereof comprises the VH CDR2 of one of theantibodies in Table 2. In certain embodiments, the antibody orantigen-binding fragment thereof comprises the VH CDR3 of one of theantibodies in Table 2. In some embodiments, the antibody orantigen-binding fragment thereof comprises one, two or all three of theVH CDRs of one of the antibodies in Table 2 (e.g., the VH CDRs in onerow of Table 2). In certain embodiments, the antibody or antigen-bindingfragment thereof comprises one, two or all three of the VL CDRs of oneof the antibodies in Table 1 (e.g., the VL CDRs in one row in Table 1).

In another embodiment, an antibody or fragment thereof described herein,which specifically binds to GITR (e.g., human GITR), comprises a lightchain variable region (VL) comprising:

(a) a VL CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence KSSQSLLNSX₁NQKNYLX₂ (SEQ ID NO: 10), wherein

X₁ is G or S

X₂ is T or S; and/or

(b) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence WASTRES (SEQ ID NO: 11); and/or

(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence QNX₁YSX₂PYT (SEQ ID NO: 12), wherein

X₁ is D or E

X₂ is Y, F or S.

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two, or all three of the VL CDRs above. Incertain embodiments, the antibody or antigen-binding fragment thereofcomprises the VL CDR1 of one of the antibodies in Table 1. In someembodiments, the antibody or antigen-binding fragment thereof comprisesthe VL CDR2 of one of the antibodies in Table 1. In certain embodiments,the antibody or antigen-binding fragment thereof comprises the VL CDR3of one of the antibodies in Table 1. In certain embodiments, theantibody or antigen-binding fragment thereof comprises one, two or allthree of the VL CDRs of one of the antibodies in Table 1 (e.g., the VLCDRs in one row of Table 1). In some embodiments, the antibody orantigen-binding fragment thereof comprises the VL framework regionsdescribed herein. In specific embodiments, the antibody orantigen-binding fragment thereof comprises the VL framework regions(FRs) of an antibody set forth in Table 3 (e.g., one, two, three, orfour of the framework regions in one row of Table 3).

In another embodiment, an antibody or fragment thereof described herein,which specifically binds to GITR (e.g., human GITR), comprises a heavychain variable region (VH) comprising:

(a) a VH CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁YX₂MX₃ (SEQ ID NO: 7), wherein

X₁ is D, E or G

X₂ is A or V

X₃ is Y or H; and/or

(b) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈(SEQ ID NO: 8), wherein

X₁ is V or L

X₂ is R, K or Q

X₃ is Y or F

X₄ is D, E or G

X₅ is V or L

X₆ is T or S

X₇ is K, R or Q

X₈ is D, E or G; and/or

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence SGTVRGFAY (SEQ ID NO: 9).

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two or all three of the VH CDRs above. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesthe VH CDR1 of one of the antibodies in Table 2. In some embodiments,the antibody or antigen-binding fragment thereof comprises the VH CDR2of one of the antibodies in Table 2. In certain embodiments, theantibody or antigen-binding fragment thereof comprises the VH CDR3 ofone of the antibodies in Table 2. In some embodiments, the antibody orantigen-binding fragment thereof comprises one, two or all three of VHCDRs of one of the antibodies in Table 2 (e.g., the VH CDRs in one rowin Table 2). In some embodiments, the antibody or antigen-bindingfragment thereof comprises one, two or all three of the VH CDRs of oneof the antibodies in Table 2. In some embodiments, the antibody orantigen-binding fragment thereof comprises the VH frameworks describedherein. In specific embodiments, the antibody or antigen-bindingfragment thereof comprises the VH framework regions of an antibody setforth in Tables 4 (e.g., one, two, three or four of the frameworkregions in one row of Table 4).

In a particular embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR), comprises:

(a) a VL CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence KSSQSLLNSX₁NQKNYLX₂ (SEQ ID NO: 10), wherein

X₁ is G or S

X₂ is T or S; and/or

(b) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence WASTRES (SEQ ID NO: 11); and/or

(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence QNX₁YSX₂PYT (SEQ ID NO: 12), wherein

X₁ is D or E

X₂ is Y, F or S.

(d) a VH CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁YX₂MX₃ (SEQ ID NO: 7), wherein

X₁ is D, E or G

X₂ is A or V

X₃ is Y or H; and/or

(e) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈ (SEQ ID NO: 8),wherein

X₁ is V or L

X₂ is R, K or Q

X₃ is Y or F

X₄ is D, E or G

X₅ is V or L

X₆ is T or S

X₇ is K, R or Q

X₈ is D, E or G; and/or

(f) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence SGTVRGFAY (SEQ ID NO: 9).

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two, three, four, five or all six of the CDRsabove. In certain embodiments, the antibody or antigen-binding fragmentthereof comprises the VL CDR1 of one of the antibodies in Table 1. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises the VL CDR2 of one of the antibodies in Table 1. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesthe VL CDR3 of one of the antibodies in Table 1. In some embodiments,the antibody or antigen-binding fragment thereof comprises the VH CDR1of one of the antibodies in Table 2. In some embodiments, the antibodyor antigen-binding fragment thereof comprises the VH CDR2 of one of theantibodies in Table 2. In certain embodiments, the antibody orantigen-binding fragment thereof comprises the VH CDR3 of one of theantibodies in Table 2. In some embodiments, the antibody orantigen-binding fragment thereof comprises one, two or all three of theVH CDRs of one of the antibodies in Table 2 (e.g., the VH CDRs in onerow in Table 2). In certain embodiments, the antibody or antigen-bindingfragment thereof comprises one, two or all three of the VL CDRs of oneof the antibodies in Table 1 (e.g., the VL CDRs in one row in Table 1).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprises alight chain variable region (VL) and a heavy chain variable region (VH),wherein

(i) the VL comprises:

(a) a VL CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence KSSQSLLNSX₁NQKNYLX₂ (SEQ ID NO: 10), wherein

X₁ is G or S

X₂ is T or S; and/or

(b) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence WASTRES (SEQ ID NO: 11); and/or

(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence QNX₁YSX₂PYT (SEQ ID NO: 12), wherein

X₁ is D or E

X₂ is Y, F or S; and

(ii) the VH comprises:

(a) a VH CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁YX₂MX₃ (SEQ ID NO: 7), wherein

X₁ is D, E or G

X₂ is A or V

X₃ is Y or H; and/or

(b) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈(SEQ ID NO: 8), wherein

X₁ is V or L

X₂ is R, K or Q

X₃ is Y or F

X₄ is D, E or G

X₅ is V or L

X₆ is T or S

X₇ is K, R or Q

X₈ is D, E or G; and/or

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence SGTVRGFAY (SEQ ID NO: 9).

In specific embodiments, the VL comprises two or all three of the VLCDRs above and/or the VH comprises two or all three of the VH CDRsabove. In certain embodiments, the antibody or antigen-binding fragmentthereof comprises the VL CDR1 of one of the antibodies in Table 1. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises the VL CDR2 of one of the antibodies in Table 1. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesthe VL CDR3 of one of the antibodies in Table 1. In some embodiments,the antibody or antigen-binding fragment thereof comprises the VH CDR1of one of the antibodies in Table 2. In some embodiments, the antibodyor antigen-binding fragment thereof comprises the VH CDR2 of one of theantibodies in Table 2. In certain embodiments, the antibody orantigen-binding fragment thereof comprises the VH CDR3 of one of theantibodies in Table 2. In some embodiments, the antibody orantigen-binding fragment thereof comprises one, two or all three of theVH CDRs of one of the antibodies in Table 2 (e.g., the VH CDRs in onerow in Table 2). In certain embodiments, the antibody or antigen-bindingfragment thereof comprises one, two or all three of the VL CDRs of oneof the antibodies in Table 1 (e.g., the VL CDRs in one row in Table 1).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprises alight chain variable region (VL) and a heavy chain variable region (VH),wherein

(i) the VL comprises:

(a) a VL CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence KSSQSLLNSX₁NQKNYLX₂ (SEQ ID NO: 10), wherein

X₁ is G or S

X₂ is T or S; and/or

(b) a VL CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence WASTRES (SEQ ID NO: 11); and/or

(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence QNX₁YSX₂PYT (SEQ ID NO: 12), wherein

X₁ is D or E

X₂ is Y, F, or S; and

(ii) the VH comprises:

(a) a VH CDR1 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁YX₂MX₃ (SEQ ID NO: 7), wherein

X₁ is D, E or G

X₂ is A or V

X₃ is Y or H; and/or

(b) a VH CDR2 comprising, consisting of, or consisting essentially ofthe amino acid sequence X₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈(SEQ ID NO: 8), wherein

X₁ is V or L

X₂ is R, K or Q

X₃ is Y or F

X₄ is D, E or G

X₅ is V or L

X₆ is T or S

X₇ is K, R or Q

X₈ is D, E or G; and/or

(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence of SGTVRGX₁X₂X₃ (SEQ ID NO: 3), wherein

X₁ is F, A, V, L, I, P, M, Y, W, H or S

X₂ is A or D

X₃ is Y, G, N, Q, S, T, C, W, F, H or V.

In certain embodiments, provided herein is an antibody or fragmentthereof which specifically binds to GITR (e.g., human GITR) andcomprises one, two or three of the light chain variable region (VL)complementarity determining regions (CDRs) of an antibody in Table 1(e.g., the VL CDRs in one row of Table 1). In some embodiments, providedherein is an antibody or fragment thereof which specifically binds toGITR (e.g., human GITR) and comprises one, two or three of the heavychain variable region (VH) CDRs of any one of any one of antibodies inTable 2 (e.g., the VH CDRs in one row of Table 2).

In certain embodiments, provided herein is an antibody or fragmentthereof which specifically binds to GITR (e.g., human GITR) andcomprises a light chain variable region (VL) comprising one, two or allthree of the VL CDRs of an antibody in Table 1 (e.g., the VL CDRs in onerow of Table 1). In some embodiments, the antibody or antigen-bindingfragment thereof comprises one, two, three or all four of the VLframework regions described herein. In specific embodiments, theantibody or antigen-binding fragment thereof comprises one, two, threeor all four of the VL framework regions (FRs) set forth in Table 3(e.g., one, two, three or four of the framework regions in one row inTable 3).

In certain embodiments, provided herein is an antibody or fragmentthereof which specifically binds to GITR (e.g., human GITR) andcomprises a heavy chain variable region (VH) comprising one, two or allthree of the VH CDRs of an antibody in Table 2 (e.g., the VH CDRs in onerow of Table 2). In some embodiments, the antibody or antigen-bindingfragment thereof comprises one, two, three or all four of the VHframework regions described herein. In specific embodiments, theantibody or antigen-binding fragment thereof comprises one, two, threeor all four of the VH framework regions (FRs) set forth in Table 4(e.g., one, two, three, or four of the framework regions in one row inTable 4).

In certain embodiments, provided herein is an antibody or fragmentthereof which specifically binds to GITR (e.g., human GITR) andcomprises light chain variable region (VL) CDRs and heavy chain variableregion (VH) CDRs of any one of antibodies Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, Hum231#1,231-32-15 or antibodies 1-107, or antibodies pab2159, pab2160, orpab2161, for example as set forth in Tables 1 and 2 (e.g., the VH CDRsand VL CDRs in the same row are all from the same antibody as designatedby the name of the antibody in the first column of Tables 1 and 2, forexample, the VL CDRs and VH CDRs in the first row of Tables land 2respectively are all from antibody 231-32-15). In some embodiments, theantibody or antigen-binding fragment thereof comprises the VL frameworkregions and VH frameworks described herein. In specific embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions (FRs) and VH framework regions set forth in Tables 3 and 4(e.g., the VL FRs and VH FRs are all from the same antibody).

In a particular embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR), comprises a light chain variable region (VL)comprising VL CDR1, VL CDR2, and VL CDR3 as set forth in Table 1, forexample, VL CDR1, VL CDR2, and VL CDR3 of any one of antibodiesHum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, Hum231#1, 231-32-15 or antibodies 1-107, or antibodiespab2159, pab2160, or pab2161, (e.g., the VL CDRs are in one row of Table1). In some embodiments, the antibody or antigen-binding fragmentthereof comprises VL framework regions of an antibody set forth in Table3 (e.g., one, two, three, or four of the framework regions in one row inTable 3). In certain embodiments, the antibody or antigen-bindingfragment thereof comprises a light chain variable region sequencecomprising one, two, three or four of the framework regions of the lightchain variable region sequence of SEQ ID NO: 204 or SEQ ID NO: 205. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises one, two, three or four of the framework regions of a lightchain variable region sequence which is at least 75%, 80%, 85%, 90%,95%, or 100% identical to one, two, three or four of the frameworkregions of a light chain variable region sequence selected from thegroup consisting of SEQ ID NO: 202, SEQ ID NO: 207, SEQ ID NO: 208, andSEQ ID NOs: 400-518. In some embodiments, the antibody orantigen-binding fragment thereof comprises one, two, three or four ofthe framework regions of a light chain variable region sequence which isat least 75%, 80%, 85%, 90%, 95%, or 100% identical to one, two, threeor four of the framework regions of the light chain variable regionsequence of SEQ ID NO: 519. In certain embodiments, an antibody orantigen-binding fragment thereof comprises a light chain variableframework region that is or is derived from an amino acid sequenceencoded by a human gene, wherein the amino acid sequence is selectedfrom the group consisting of IGKV4-1*01 (SEQ ID NO: 607) and IGKV3-7*02(SEQ ID NO: 608). In specific embodiments, the light chain variableframework region that is derived from said amino acid sequence consistsof said amino acid sequence but for the presence of up to 10 amino acidsubstitutions, deletions, and/or insertions, preferably up to 10 aminoacid substitutions. In a particular embodiment, the light chain variableframework region that is derived from said amino acid sequence consistsof said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aminoacid residues being substituted for an amino acid found in an analogousposition in a corresponding non-human light chain variable frameworkregion. In some embodiments, an antibody or antigen-binding fragmentthereof comprises a light chain variable framework region that isderived from amino acid sequence SEQ ID NO: 607 or SEQ ID NO: 608,wherein at least one amino acid in amino acid sequence SEQ ID NO: 607 orSEQ ID NO: 608 is substituted with an amino acid in an analogousposition in a corresponding non-human light chain variable frameworkregion. In a specific embodiment, the amino acid substitution is atamino acid position 87, wherein the amino acid position is indicatedaccording to the Kabat numbering. In particular embodiments, the aminoacid substitution is 87H, wherein the amino acid position is indicatedaccording to the Kabat numbering.

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of Hum231#1, for example, the VL CDR1,VL CDR2, and VL CDR3 of Hum231#1 as set forth in Table 1 (SEQ ID NOS:16, 17, and 18, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVL framework regions derived from the VL of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VL framework regionsderived from a human light chain variable kappa subfamily. In someembodiments, the antibody or antigen-binding fragment thereof comprisesVL framework regions of an antibody set forth in Table 3 (e.g., theframework regions of Hum231#1).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of Hum231#2, for example, the VL CDR1,VL CDR2, and VL CDR3 of Hum231#2 as set forth in Table 1 (SEQ ID NOS:16, 17, and 18, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVL framework regions derived from the VL of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VL framework regionsderived from a human light chain variable kappa subfamily. In someembodiments, the antibody or antigen-binding fragment thereof comprisesVL framework regions of an antibody set forth in Table 3 (e.g., theframework regions of Hum231#2).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1964, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1964 as set forth in Table 1 (SEQ ID NOS:101, 105, and 106, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1964).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of A pab1965, for example, the VLCDR1, VL CDR2, and VL CDR3 of pab1965 as set forth in Table 1 (SEQ IDNOS: 102, 105, and 107, respectively). In certain embodiments, theantibody or antigen-binding fragment further comprises one, two, threeor all four VL framework regions derived from the VL of a human orprimate antibody. In specific embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVL framework regions derived from a human light chain variable kappasubfamily. In some embodiments, the antibody or antigen-binding fragmentthereof comprises VL framework regions of an antibody set forth in Table3 (e.g., the framework regions of pab1965).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1966, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1966 as set forth in Table 1 (SEQ ID NOS:102, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1966).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1967, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1967 as set forth in Table 1 (SEQ ID NOS:103, 105, and 108, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1967).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1968, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1968 as set forth in Table 1 (SEQ ID NOS:101, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1968).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1969, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1969 as set forth in Table 1 (SEQ ID NOS:103, 105, and 109, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1969).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1970, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1970 as set forth in Table 1 (SEQ ID NOS:101, 105, and 109, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1970).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1971, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1971 as set forth in Table 1 (SEQ ID NOS:103, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1971).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1972, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1972 as set forth in Table 1 (SEQ ID NOS:104, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1972).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1973, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1973 as set forth in Table 1 (SEQ ID NOS:103, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1973).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1975, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1975 as set forth in Table 1 (SEQ ID NOS:102, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1975).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1976, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1976 as set forth in Table 1 (SEQ ID NOS:101, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1976).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1977, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1977 as set forth in Table 1 (SEQ ID NOS:103, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1977).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1979, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1979 as set forth in Table 1 (SEQ ID NOS:102, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1979).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1980, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1980 as set forth in Table 1 (SEQ ID NOS:101, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1980).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1981, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1981 as set forth in Table 1 (SEQ ID NOS:103, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1981).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab1983, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab1983 as set forth in Table 1 (SEQ ID NOS:102, 105, and 107, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab1983).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab2159, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab2159 as set forth in Table 1 (SEQ ID NOS:102, 105, and 109, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab2159). In a particular embodiment, anantibody or fragment thereof described herein, which specifically bindsto GITR (e.g., human GITR), comprises the VL CDR1, VL CDR2, and VL CDR3of pab2160, for example, the VL CDR1, VL CDR2, and VL CDR3 of pab2160 asset forth in Table 1 (SEQ ID NOS: 102, 105, and 107, respectively). Incertain embodiments, the antibody or antigen-binding fragment furthercomprises one, two, three or all four VL framework regions derived fromthe VL of a human or primate antibody. In specific embodiments, theantibody or antigen-binding fragment further comprises one, two, threeor all four VL framework regions derived from a human light chainvariable kappa subfamily. In some embodiments, the antibody orantigen-binding fragment thereof comprises VL framework regions of anantibody set forth in Table 3 (e.g., the framework regions of pab2160).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, and VL CDR3 of pab2161, for example, the VL CDR1,VL CDR2, and VL CDR3 of pab2161 as set forth in Table 1 (SEQ ID NOS:103, 105, and 109, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody. In specific embodiments, the antibody or antigen-bindingfragment further comprises one, two, three or all four VL frameworkregions derived from a human light chain variable kappa subfamily. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises VL framework regions of an antibody set forth in Table 3(e.g., the framework regions of pab2161).

In a particular embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR), comprises a heavy chain variable region (VH)comprising VH CDR1, VH CDR2, and VH CDR3 as set forth in Table 2, forexample, VH CDR1, VH CDR2, and VH CDR3 of any one of antibodiesHum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15, or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161, (e.g., the VH CDRs in one rowin Table 2). In some embodiments, the antibody or antigen-bindingfragment thereof comprises VH framework regions of an antibody set forthin Table 4 (e.g., one, two, three, or four of the framework regions inone row in Table 4). In certain embodiments, the antibody orantigen-binding fragment thereof comprises one, two, three or all fourof the framework regions of the heavy chain variable region sequence ofSEQ ID NO: 203. In some embodiments, the antibody or antigen-bindingfragment thereof comprises one, two, three, or four of the frameworkregions of a heavy chain variable region sequence which is at least 75%,80%, 85%, 90%, 95% or 100% identical to one, two, three or four of theframework regions of a heavy chain variable region sequence selectedfrom the group consisting of SEQ ID NO: 201, SEQ ID NO: 206, and SEQ IDNOS: 215 to 389. In certain embodiments, the antibody or antigen-bindingfragment thereof comprises a heavy chain variable framework region thatis or is derived from an amino acid sequence encoded by a human gene,wherein the amino acid sequence is selected from the group consisting ofIGHV1-2*02 (SEQ ID NO: 601), IGHV1-3*01 (SEQ ID NO: 602), IGHV1-46*01(SEQ ID NO: 603), IGHV1-18*01 (SEQ ID NO: 604), IGHV1-69*01 (SEQ ID NO:605), and IGHV7-4-1*02 (SEQ ID NO: 606). In specific embodiments, theheavy chain variable framework region that is derived from said aminoacid sequence consists of said amino acid sequence but for the presenceof up to 10 amino acid substitutions, deletions, and/or insertions,preferably up to 10 amino acid substitutions. In a particularembodiment, the heavy chain variable framework region that is derivedfrom said amino acid sequence consists of said amino acid sequence with1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues being substitutedfor an amino acid found in an analogous position in a correspondingnon-human heavy chain variable framework region. In specificembodiments, the antibody or antigen-binding fragment thereof comprisesa heavy chain variable framework region that is derived from amino acidsequence SEQ ID NO: 601, wherein at least one amino acid of amino acidsequence SEQ ID NO: 601 is substituted with an amino acid in ananalogous position in a corresponding non-human heavy chain variableframework region. In certain embodiments, the amino acid substitution isat an amino acid position selected from the group consisting of 24, 48,67, 71, 73, and 94, wherein the amino acid position of each group memberis indicated according to the Kabat numbering. In specific embodiments,the amino acid substitution selected from the group consisting of 24G,48I, 67A, 71V, 73K, and 94K, wherein the amino acid position of eachgroup member is indicated according to the Kabat numbering.

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of Hum231#1, for example, the VH CDR1,VH CDR2, and VH CDR3 of Hum231#1 as set forth in Table 2 (SEQ ID NOS:13, 14, and 15, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of Hum231#1).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of Hum231#2, for example, the VH CDR1,VH CDR2, and VH CDR3 of Hum231#2 as set forth in Table 2 (SEQ ID NOS:13, 14, and 15, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of Hum231#2).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1964, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1964 as set forth in Table 2 (SEQ ID NOS: 19,24, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1964).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1965, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1965 as set forth in Table 2 (SEQ ID NOS: 19,25, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1965).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1966, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1966 as set forth in Table 2 (SEQ ID NOS: 19,26, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1966).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1967, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1967 as set forth in Table 2 (SEQ ID NOS: 20,27, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1967).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1968, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1968 as set forth in Table 2 (SEQ ID NOS: 21,28, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1968).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1969, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1969 as set forth in Table 2 (SEQ ID NOS: 22,29, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1969).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1970, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1970 as set forth in Table 2 (SEQ ID NOS: 21,24, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1970).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1971, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1971 as set forth in Table 2 (SEQ ID NOS: 21,177, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1971).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1972, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1972 as set forth in Table 2 (SEQ ID NOS: 23,31, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1972).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1973, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1973 as set forth in Table 2 (SEQ ID NOS: 19,32, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1973).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1975, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1975 as set forth in Table 2 (SEQ ID NOS: 22,29, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1975).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1976, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1976 as set forth in Table 2 (SEQ ID NOS: 22,29, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1976).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1977, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1977 as set forth in Table 2 (SEQ ID NOS: 22,29, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1977).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1979, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1979 as set forth in Table 2 (SEQ ID NOS: 22,33, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1979).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1980, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1980 as set forth in Table 2 (SEQ ID NOS: 22,33, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1980).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1981, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1981 as set forth in Table 2 (SEQ ID NOS: 22,33, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1981).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab1983, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab1983 as set forth in Table 2 (SEQ ID NOS: 19,24, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab1983).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab2159, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab2159 as set forth in Table 2 (SEQ ID NOS: 19,144, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab2159).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab2160, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab2160 as set forth in Table 2 (SEQ ID NOS:119, 162, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab2160).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VH CDR1, VH CDR2, and VH CDR3 of pab2161, for example, the VH CDR1,VH CDR2, and VH CDR3 of pab2161 as set forth in Table 2 (SEQ ID NOS: 22,121, and 34, respectively). In certain embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourVH framework regions derived from the VH of a human or primate antibody.In specific embodiments, the antibody or antigen-binding fragmentfurther comprises one, two, three or all four VH framework regionsderived from a human heavy chain variable subfamily (e.g., one ofsubfamilies 1 to 7). In some embodiments, the antibody orantigen-binding fragment thereof comprises VH framework regions of anantibody set forth in Table 4 (e.g., the framework regions of pab2161).

In a particular embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR), comprises (i) a heavy chain variable region (VH)comprising VH CDR1, VH CDR2, and VH CDR3 as set forth in Table 2, forexample, VH CDR1, VH CDR2, and VH CDR3 of any one of antibodiesHum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161, (e.g. the VH CDRs in one row inTable 2), and (ii) a light chain variable region (VL) comprising VLCDR1, VL CDR2, and VL CDR3 as set forth in Table 1, for example, VLCDR1, VL CDR2, and VL CDR3 of any one of antibodies Hum231#1, Hum231#2,pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971,pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981,pab1983, 231-32-15 or antibodies 1-107, or antibodies pab2159, pab2160,or pab2161, (e.g., the VL CDRs in one row in Table 1). In someembodiments, the antibody or antigen-binding fragment thereof comprisesVL framework regions and VH framework regions of an antibody set forthin Tables 3 and 4, respectively (e.g., the framework regions of a singleantibody as designated by its name, for example, all of the FRs are fromHum231#1 or Hum231#2).

In some embodiments, the antibody or antigen-binding fragment thereofdescribed herein comprises one, two, three or four framework regions ofa heavy chain variable region sequence which is at least 75%, 80%, 85%,90%, 95% or 100% identical to one, two, three or four of the frameworkregions of a heavy chain variable region sequence selected from thegroup consisting of SEQ ID NO: 201, SEQ ID NO: 206, and SEQ ID NOS: 215to 389. In certain embodiments, the antibody or antigen-binding fragmentthereof described herein comprises a heavy chain variable frameworkregion that is or is derived from an amino acid sequence encoded by ahuman gene, wherein the amino acid sequence is selected from the groupconsisting of IGHV1-2*02 (SEQ ID NO: 601), IGHV1-3*01 (SEQ ID NO: 602),IGHV1-46*01 (SEQ ID NO: 603), IGHV1-18*01 (SEQ ID NO: 604), IGHV1-69*01(SEQ ID NO: 605), and IGHV7-4-1*02 (SEQ ID NO: 606). In specificembodiments, the heavy chain variable framework region that is derivedfrom said amino acid sequence consists of said amino acid sequence butfor the presence of up to 10 amino acid substitutions, deletions, and/orinsertions, preferably up to 10 amino acid substitutions. In aparticular embodiment, the heavy chain variable framework region that isderived from said amino acid sequence consists of said amino acidsequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues beingsubstituted for an amino acid found in an analogous position in acorresponding non-human heavy chain variable framework region. Inspecific embodiments, the antibody or antigen-binding fragment thereofdescribed herein comprises a heavy chain variable framework region thatis derived from amino acid sequence SEQ ID NO: 601, wherein at least oneamino acid in amino acid sequence SEQ ID NO: 601 is substituted with anamino acid in an analogous position in a corresponding non-human lightchain variable framework region. In certain embodiments, the amino acidsubstitution is at an amino acid position selected from the groupconsisting of 24, 48, 67, 71, 73, and 94, wherein the amino acidposition of each group member is indicated according to the Kabatnumbering. In specific embodiments, the amino acid substitution isselected from the group consisting of 24G, 48I, 67A, 71V, 73K, and 94K,wherein the amino acid position of each group member is indicatedaccording to the Kabat numbering.

In some embodiments, the antibody or antigen-binding fragment thereofdescribed herein comprises VL framework regions of an antibody set forthin Table 3. In certain embodiments, the antibody or antigen-bindingfragment thereof described herein comprises a light chain variableregion sequence comprising one, two, three or four of the frameworkregions of the light chain variable region sequence of SEQ ID NO: 204 orSEQ ID NO: 205. In some embodiments, the antibody or antigen-bindingfragment thereof described herein comprises one, two, three or fourframework regions of a light chain variable region sequence which is atleast 75%, 80%, 85%, 90%, 95%, or 100% identical to one, two, three orfour of the framework regions of a light chain variable region sequenceselected from the group consisting of SEQ ID NO: 202, SEQ ID NO: 207,SEQ ID NO: 208, and SEQ ID NOs: 400-518. In some embodiments, theantibody or antigen-binding fragment thereof described herein comprisesone, two, three or four framework regions of a light chain variableregion sequence which is at least 75%, 80%, 85%, 90%, 95%, or 100%identical to one, two, three or four of the framework regions of thelight chain variable region sequence of SEQ ID NO: 519. In certainembodiments, an antibody or antigen-binding fragment thereof describedherein comprises a light chain variable framework region that is or isderived from an amino acid sequence encoded by a human gene, wherein theamino acid sequence is selected from the group consisting of IGKV4-1*01(SEQ ID NO: 607) and IGKV3-7*02 (SEQ ID NO: 608). In specificembodiments, the light chain variable framework region that is derivedfrom said amino acid sequence consists of said amino acid sequence butfor the presence of up to 10 amino acid substitutions, deletions, and/orinsertions, preferably up to 10 amino acid substitutions. In aparticular embodiment, the light chain variable framework region that isderived from said amino acid sequence consists of said amino acidsequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues beingsubstituted for an amino acid found in an analogous position in acorresponding non-human light chain variable framework region. In someembodiments, an antibody or antigen-binding fragment thereof describedherein comprises a light chain variable framework region that is derivedfrom amino acid sequence SEQ ID NO: 607 or SEQ ID NO: 608, wherein atleast one amino acid of amino acid sequence SEQ ID NO: 607 or SEQ ID NO:608 with an amino acid in an analogous position in a correspondingnon-human light chain variable framework region. In a specificembodiment, the amino acid substitution is at amino acid position 87,wherein the amino acid position is indicated according to the Kabatnumbering. In particular embodiments, the amino acid substitution is87H, wherein the amino acid position is indicated according to the Kabatnumbering.

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 ofHum231#1, for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2,and VH CDR3 of Hum231#1 as set forth in Tables 1 and 2 (SEQ ID NOS: 16,17, 18, 13, 14, and 15, respectively). In certain embodiments, theantibody or antigen-binding fragment further comprises one, two, threeor all four VL framework regions derived from the VH of a human orprimate antibody and one, two, three or all four VH framework regionsderived from the VH of a human or primate antibody. In some embodiments,the antibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of Hum231#1).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 ofHum231#2, for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2,and VH CDR3 of Hum231#2 as set forth in Tables 1 and 2 (SEQ ID NOS: 16,17, 18, 13, 14, and 15, respectively). In certain embodiments, theantibody or antigen-binding fragment further comprises one, two, threeor all four VL framework regions derived from the VL of a human orprimate antibody and one, two, three or all four VH framework regionsderived from the VH of a human or primate antibody. In some embodiments,the antibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the frameworks of a single antibody asdesignated by its name, for example, the framework regions of Hum231#1or Hum231#2).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1964,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1964 as set forth in Tables 1 and 2 (SEQ ID NOS: 101, 105,106, 19, 24, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1964).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1965,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1965 as set forth in Tables 1 and 2 (SEQ ID NOS: 102, 105,107, 19, 25, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1965).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1966,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1966 as set forth in Tables 1 and 2 (SEQ ID NOS: 102, 105,107, 19, 26, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1966).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1967,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1967 as set forth in Tables 1 and 2 (SEQ ID NOS: 103, 105,108, 20, 27, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1967).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1968,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1968 as set forth in Tables 1 and 2 (SEQ ID NOS: 101, 105,107, 21, 28, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1968).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1969,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1969 as set forth in Tables 1 and 2 (SEQ ID NOS: 103, 105,109, 22, 29, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1969).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1970,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1970 as set forth in Tables 1 and 2 (SEQ ID NOS: 101, 105,109, 21, 24, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1970).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1971,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1971 as set forth in Tables 1 and 2 (SEQ ID NOS: 103, 105,107, 21, 177, and 34, respectively). In certain embodiments, theantibody or antigen-binding fragment further comprises one, two, threeor all four VL framework regions derived from the VL of a human orprimate antibody and one, two, three or all four VH framework regionsderived from the VH of a human or primate antibody. In some embodiments,the antibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1971).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1972,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1972 as set forth in Tables 1 and 2 (SEQ ID NOS: 104, 105,107, 23, 31, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1972).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1973,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1973 as set forth in Tables 1 and 2 (SEQ ID NOS: 103, 105,107, 19, 32, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1973).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1975,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1975 as set forth in Tables 1 and 2 (SEQ ID NOS: 102, 105,107, 22, 29, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1975).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1976,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1976 as set forth in Tables 1 and 2 (SEQ ID NOS: 101, 105,107, 22, 29, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1976).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1977,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1977 as set forth in Tables 1 and 2 (SEQ ID NOS: 103, 105,107, 22, 29, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1977).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1979,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1979 as set forth in Tables 1 and 2 (SEQ ID NOS: 102, 105,107, 22, 33, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1979).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1980,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1980 as set forth in Tables 1 and 2 (SEQ ID NOS: 101, 105,107, 22, 33, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1980).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1981,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1981 as set forth in Tables 1 and 2 (SEQ ID NOS: 103, 105,107, 22, 33, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1981).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab1983,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab1983 as set forth in Tables 1 and 2 (SEQ ID NOS: 102, 105,107, 19, 24, and 34, respectively). In certain embodiments, the antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from the VL of a human or primateantibody and one, two, three or all four VH framework regions derivedfrom the VH of a human or primate antibody. In some embodiments, theantibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab1983).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab2159,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab2159 as set forth in Tables 1 and 2 (SEQ ID NOS: 102, 105,109, 19, 144, and 34, respectively). In certain embodiments, theantibody or antigen-binding fragment further comprises one, two, threeor all four VL framework regions derived from the VL of a human orprimate antibody and one, two, three or all four VH framework regionsderived from the VH of a human or primate antibody. In some embodiments,the antibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab2159).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab2160,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab2160 as set forth in Tables 1 and 2 (SEQ ID NOS: 102, 105,107, 119, 162, and 34, respectively). In certain embodiments, theantibody or antigen-binding fragment further comprises one, two, threeor all four VL framework regions derived from the VL of a human orprimate antibody and one, two, three or all four VH framework regionsderived from the VH of a human or primate antibody. In some embodiments,the antibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab2160).

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of pab2161,for example, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 of pab2161 as set forth in Tables 1 and 2 (SEQ ID NOS: 103, 105,109, 22, 121, and 34, respectively). In certain embodiments, theantibody or antigen-binding fragment further comprises one, two, threeor all four VL framework regions derived from the VL of a human orprimate antibody and one, two, three or all four VH framework regionsderived from the VH of a human or primate antibody. In some embodiments,the antibody or antigen-binding fragment thereof comprises VL frameworkregions and VH framework regions of an antibody set forth in Tables 3and 4, respectively (e.g., the framework regions of pab2161).

In certain embodiments, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising the amino acid sequence of a VL domain of an antibody listedin FIG. 23 or any one of FIGS. 24A-24C (e.g., the VL domain in one rowof FIG. 23 or any one of FIGS. 24A-24C). In certain embodiments, anantibody or fragment thereof that specifically binds to GITR (e.g.,human GITR) comprises a VL domain comprising the amino acid sequence ofa VL domain of an antibody listed in Table 17 (e.g., the VL domain inone row of Table 17). In a specific embodiment, an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprises aVL domain comprising SEQ ID NO: 207 (e.g., antibody Hum231#1). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain comprising SEQ IDNO: 208 (e.g., antibody Hum231#2). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 435 (e.g., antibodypab1964). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 437 (e.g., antibody pab1965). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 440(e.g., antibody pab1966). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 441 (e.g., antibodypab1967). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 444 (e.g., antibody pab1968). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 458(e.g., antibody pab1969). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 459 (e.g., antibodypab1970). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 453 (e.g., antibody pab1971). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 463(e.g., antibody pab1972). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 519 (e.g., antibodypab1973). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 440 (e.g., antibody pab1975). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 444(e.g., antibody pab1976). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 453 (e.g., antibodypab1977). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 440 (e.g., antibody pab1979). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 444(e.g., antibody pab1980). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 453 (e.g., antibodypab1981). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 440 (e.g., antibody pab1983). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 408(e.g., antibody pab2159). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 423 (e.g., antibodypab2160). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 486 (e.g., antibody pab2161).

In some embodiments, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain consisting of orconsisting essentially of the amino acid sequence of a VL domain of anantibody listed in FIG. 23 or any one of FIGS. 24A-24C (e.g., the VLdomain in one row of FIG. 23 or any one of FIGS. 24A-24C). In someembodiments, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain consisting of orconsisting essentially of the amino acid sequence of a VL domain of anantibody listed in Table 17 (e.g., the VL domain in one row of Table17). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domainconsisting of or consisting essentially of SEQ ID NO: 207 (e.g.,antibody Hum231#1). In a specific embodiment, an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprises aVL domain consisting of or consisting essentially of SEQ ID NO: 208(e.g., antibody Hum231#2). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 435 (e.g., antibody pab1964). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 437 (e.g., antibody pab1965). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 440 (e.g., antibody pab1966). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 441 (e.g., antibody pab1967). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 444 (e.g., antibody pab1968). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 458 (e.g., antibodypab1969). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domainconsisting of or consisting essentially of SEQ ID NO: 459 (e.g.,antibody pab1970). In a specific embodiment, an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprises aVL domain consisting of or consisting essentially of SEQ ID NO: 453(e.g., antibody pab1971). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 463 (e.g., antibody pab1972). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 519 (e.g., antibody pab1973). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 440 (e.g., antibody pab1975). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 444 (e.g., antibody pab1976). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 453 (e.g., antibody pab1977). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 440 (e.g., antibody pab1979). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 444 (e.g., antibody pab1980). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 453 (e.g., antibody pab1981). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 440 (e.g., antibody pab1983). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 408 (e.g., antibody pab2159). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 423 (e.g., antibody pab2160). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain consisting of or consisting essentially of SEQ IDNO: 486 (e.g., antibody pab2161).

In certain embodiments, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domaincomprising the amino acid sequence of a VH domain of an antibody listedin FIG. 23 or any one of FIGS. 24A-24C (e.g., the VH domain in one rowin FIG. 23 or any one of FIGS. 24A-24C). In certain embodiments, anantibody or fragment thereof that specifically binds to GITR (e.g.,human GITR) comprises a VH domain comprising the amino acid sequence ofa VH domain of an antibody listed in Table 17 (e.g., the VH domain inone row in Table 17). In a specific embodiment, an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprises aVH domain comprising SEQ ID NO: 206 (e.g., antibody Hum231#1). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VH domain comprising SEQ IDNO: 206 (e.g., antibody Hum231#2). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain comprising SEQ ID NO: 249 (e.g., antibodypab1964). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domaincomprising SEQ ID NO: 251 (e.g., antibody pab1965). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VH domain comprising SEQ ID NO: 254(e.g., antibody pab1966). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain comprising SEQ ID NO: 255 (e.g., antibodypab1967). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domaincomprising SEQ ID NO: 259 (e.g., antibody pab1968). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VH domain comprising SEQ ID NO: 276(e.g., antibody pab1969). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain comprising SEQ ID NO: 277 (e.g., antibodypab1970). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domaincomprising SEQ ID NO: 280 (e.g., antibody pab1971). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VH domain comprising SEQ ID NO: 284(e.g., antibody pab1972). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain comprising SEQ ID NO: 304 (e.g., antibodypab1973). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domaincomprising SEQ ID NO: 276 (e.g., antibody pab1975). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VH domain comprising SEQ ID NO: 276(e.g., antibody pab1976). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain comprising SEQ ID NO: 276 (e.g., antibodypab1977). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domaincomprising SEQ ID NO: 345 (e.g., antibody pab1979). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VH domain comprising SEQ ID NO: 345(e.g., antibody pab1980). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain comprising SEQ ID NO: 345 (e.g., antibodypab1981). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domaincomprising SEQ ID NO: 249 (e.g., antibody pab1983). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VH domain comprising SEQ ID NO: 224(e.g., antibody pab2159). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain comprising SEQ ID NO: 237 (e.g., antibodypab2160). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domaincomprising SEQ ID NO: 315 (e.g., antibody pab2161).

In some embodiments, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VH domain consisting of orconsisting essentially of the amino acid sequence of a VH domain of anantibody listed in FIG. 23 or any one of FIGS. 24A-24C (e.g., the VHdomain in one row in FIG. 23 or any one of FIGS. 24A-24C). In someembodiments, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VH domain consisting of orconsisting essentially of the amino acid sequence of a VH domain of anantibody listed in Table 17 (e.g., the VH domain in one row in Table17). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domainconsisting of or consisting essentially of SEQ ID NO: 206 (e.g.,antibody Hum231#1). In a specific embodiment, an antibody or fragmentthereof that specifically binds to GITR (e.g., human GITR) comprises aVH domain consisting of or consisting essentially of SEQ ID NO: 206(e.g., antibody Hum231#2). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 249 (e.g., antibody pab1964). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 251 (e.g., antibody pab1965). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 254 (e.g., antibody pab1966). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 255 (e.g., antibody pab1967). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 259 (e.g., antibody pab1968). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 276 (e.g., antibody pab1969). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 277 (e.g., antibody pab1970). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 280 (e.g., antibody pab1971). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 284 (e.g., antibody pab1972). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 304 (e.g., antibody pab1973). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 276 (e.g., antibody pab1975). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 276 (e.g., antibody pab1976). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 276 (e.g., antibody pab1977). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 345 (e.g., antibody pab1979). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 345 (e.g., antibody pab1980). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 345 (e.g., antibody pab1981). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 249 (e.g., antibody pab1983). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 224 (e.g., antibody pab2159). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 237 (e.g., antibody pab2160). In a specific embodiment, an antibodyor fragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VH domain consisting of or consisting essentially of SEQ IDNO: 315 (e.g., antibody pab2161).

In certain embodiments, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domain anda VL domain, wherein the VH domain and the VL domain comprise the aminoacid sequence of a VH domain and a VL domain of an antibody listed inFIG. 23 or any one of FIGS. 24A-24C (e.g., the VH domain and VL domainin one row of FIG. 23 or any one of FIGS. 24A-24C). In certainembodiments, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VH domain and a VL domain, whereinthe VH domain and the VL domain comprise the amino acid sequence of a VHdomain and a VL domain of an antibody listed in Table 17 (e.g., the VHdomain and VL domain in one row of Table 17). In a specific embodiment,an antibody or fragment thereof that specifically binds to GITR (e.g.,human GITR) comprises a VL domain comprising SEQ ID NO: 207 and a VHdomain comprising SEQ ID NO: 206 (e.g., antibody Hum231#1). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain comprising SEQ IDNO: 208 and a VH domain comprising SEQ ID NO: 206 (e.g., antibodyHum231#2). In a specific embodiment, an antibody or fragment thereofthat specifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 435 and a VH domain comprising SEQ ID NO: 249(e.g., antibody pab1964). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 437 and a VH domaincomprising SEQ ID NO: 251 (e.g., antibody pab1965). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 440and a VH domain comprising SEQ ID NO: 254 (e.g., antibody pab1966). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain comprising SEQ IDNO: 441 and a VH domain comprising SEQ ID NO: 255 (e.g., antibodypab1967). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 444 and a VH domain comprising SEQ ID NO: 259(e.g., antibody pab1968). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 458 and a VH domaincomprising SEQ ID NO: 276 (e.g., antibody pab1969). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 459and a VH domain comprising SEQ ID NO: 277 (e.g., antibody pab1970). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain comprising SEQ IDNO: 453 and a VH domain comprising SEQ ID NO: 280 (e.g., antibodypab1971). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 463 and a VH domain comprising SEQ ID NO: 284(e.g., antibody pab1972). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 519 and a VH domaincomprising SEQ ID NO: 304 (e.g., antibody pab1973). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 440and a VH domain comprising SEQ ID NO: 276 (e.g., antibody pab1975). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain comprising SEQ IDNO: 444 and a VH domain comprising SEQ ID NO: 276 (e.g., antibodypab1976). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 453 and a VH domain comprising SEQ ID NO: 276(e.g., antibody pab1977). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 440 and a VH domaincomprising SEQ ID NO: 345 (e.g., antibody pab1979). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 444and a VH domain comprising SEQ ID NO: 345 (e.g., antibody pab1980). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain comprising SEQ IDNO: 453 and a VH domain comprising SEQ ID NO: 345 (e.g., antibodypab1981). In a specific embodiment, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VL domaincomprising SEQ ID NO: 440 and a VH domain comprising SEQ ID NO: 249(e.g., antibody pab1983). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain comprising SEQ ID NO: 408 and a VH domaincomprising SEQ ID NO: 224 (e.g., antibody pab2159). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain comprising SEQ ID NO: 423and a VH domain comprising SEQ ID NO: 237 (e.g., antibody pab2160). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain comprising SEQ IDNO: 486 and a VH domain comprising SEQ ID NO: 315 (e.g., antibodypab2161).

In certain embodiments, an antibody or fragment thereof thatspecifically binds to GITR (e.g., human GITR) comprises a VH domain anda VL domain, wherein the VH domain and the VL domain consist of orconsist essentially of the amino acid sequence of a VH domain and a VLdomain of an antibody listed in FIG. 23 or any one of FIGS. 24A-24C(e.g., the VH domain and VL domain in one row of FIG. 23 or any one ofFIGS. 24A-24C). In certain embodiments, an antibody or fragment thereofthat specifically binds to GITR (e.g., human GITR) comprises a VH domainand a VL domain, wherein the VH domain and the VL domain consist of orconsist essentially of the amino acid sequence of a VH domain and a VLdomain of an antibody listed in Table 17 (e.g., the VH domain and VLdomain in one row of Table 17). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 207 and the VH domain consistingof or consisting essentially of SEQ ID NO: 206 (e.g., antibodyHum231#1). In a specific embodiment, an antibody or fragment thereofthat specifically binds to GITR (e.g., human GITR) comprises a VL domainand a VH domain, wherein the VL domain consists of or consistsessentially of SEQ ID NO: 208 and the VH domain consists of or consistsessentially of SEQ ID NO: 206 (e.g., antibody Hum231#2). In a specificembodiment, an antibody or fragment thereof that specifically binds toGITR (e.g., human GITR) comprises a VL domain and a VH domain, whereinthe VL domain consists of or consists essentially of SEQ ID NO: 435 andthe VH domain consists of or consists essentially of SEQ ID NO: 249(e.g., antibody pab1964). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 437 and the VH domain consists ofor consists essentially of SEQ ID NO: 251 (e.g., antibody pab1965). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain and a VH domain,wherein the VL domain consists of or consists essentially of SEQ ID NO:440 and the VH domain consists of or consists essentially of SEQ ID NO:254 (e.g., antibody pab1966). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 441 and the VH domain consists ofor consists essentially of SEQ ID NO: 255 (e.g., antibody pab1967). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain and a VH domain,wherein the VL domain consists of or consists essentially of SEQ ID NO:444 and the VH domain consists of or consists essentially of SEQ ID NO:259 (e.g., antibody pab1968). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 458 and the VH domain consists ofor consists essentially of SEQ ID NO: 276 (e.g., antibody pab1969). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain and a VH domain,wherein the VL domain consists of or consists essentially of SEQ ID NO:459 and the VH domain consists of or consists essentially of SEQ ID NO:277 (e.g., antibody pab1970). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 453 and the VH domain consists ofor consists essentially of SEQ ID NO: 280 (e.g., antibody pab1971). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain and a VH domain,wherein the VL domain consists of or consists essentially of SEQ ID NO:463 and the VH domain consists of or consists essentially of SEQ ID NO:284 (e.g., antibody pab1972). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 519 and the VH domain consists ofor consists essentially of SEQ ID NO: 304 (e.g., antibody pab1973). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain and a VH domain,wherein the VL domain consists of or consists essentially of SEQ ID NO:440 and the VH domain consists of or consists essentially of SEQ ID NO:276 (e.g., antibody pab1975). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 444 and the VH domain consists ofor consists essentially of SEQ ID NO: 276 (e.g., antibody pab1976). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain and a VH domain,wherein the VL domain consists of or consists essentially of SEQ ID NO:453 and the VH domain consists of or consists essentially of SEQ ID NO:276 (e.g., antibody pab1977). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 440 and the VH domain consists ofor consists essentially of SEQ ID NO: 345 (e.g., antibody pab1979). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain and a VH domain,wherein the VL domain consists of or consists essentially of SEQ ID NO:444 and the VH domain consists of or consists essentially of SEQ ID NO:345 (e.g., antibody pab1980). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 453 and the VH domain consists ofor consists essentially of SEQ ID NO: 345 (e.g., antibody pab1981). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain and a VH domain,wherein the VL domain consists of or consists essentially of SEQ ID NO:440 and the VH domain consists of or consists essentially of SEQ ID NO:249 (e.g., antibody pab1983). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 408 and the VH domain consists ofor consists essentially of SEQ ID NO: 224 (e.g., antibody pab2159). In aspecific embodiment, an antibody or fragment thereof that specificallybinds to GITR (e.g., human GITR) comprises a VL domain and a VH domain,wherein the VL domain consists of or consists essentially of SEQ ID NO:423 and the VH domain consists of or consists essentially of SEQ ID NO:237 (e.g., antibody pab2160). In a specific embodiment, an antibody orfragment thereof that specifically binds to GITR (e.g., human GITR)comprises a VL domain and a VH domain, wherein the VL domain consists ofor consists essentially of SEQ ID NO: 486 and the VH domain consists ofor consists essentially of SEQ ID NO: 315 (e.g., antibody pab2161).

In certain aspects, an antibody described herein may be described by itsVL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, orits 3 VH CDRs alone. See, for example, Rader C et al., (1998) PNAS 95:8910-8915, which is incorporated herein by reference in its entirety,describing the humanization of the mouse anti-αvβ3 antibody byidentifying a complementing light chain or heavy chain, respectively,from a human light chain or heavy chain library, resulting in humanizedantibody variants having affinities as high or higher than the affinityof the original antibody. See also, Clackson T et al., (1991) Nature352: 624-628, which is incorporated herein by reference in its entirety,describing methods of producing antibodies that bind a specific antigenby using a specific VL domain (or VH domain) and screening a library forthe complementary variable domains. The screen produced 14 new partnersfor a specific VH domain and 13 new partners for a specific VL domain,which were strong binders, as determined by ELISA. See also, Kim S J &Hong H J, (2007) J Microbiol 45: 572-577, which is incorporated hereinby reference in its entirety, describing methods of producing antibodiesthat bind a specific antigen by using a specific VH domain and screeninga library (e.g., human VL library) for complementary VL domains; theselected VL domains in turn could be used to guide selection ofadditional complementary (e.g., human) VH domains.

In certain aspects, the CDRs of an antibody can be determined accordingto the Chothia numbering scheme, which refers to the location ofimmunoglobulin structural loops (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817;Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No.7,709,226). Typically, when using the Kabat numbering convention, theChothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33,or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids95 to 102, while the Chothia CDR-L1 loop is present at light chain aminoacids 24 to 34, the Chothia CDR-L2 loop is present at light chain aminoacids 50 to 56, and the Chothia CDR-L3 loop is present at light chainamino acids 89 to 97. The end of the Chothia CDR-HI loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34).

In certain aspects, provided herein are antibodies or fragments thereofthat specifically bind to GITR (e.g., human GITR) and comprise one ormore Chothia VL CDRs of a VL of any one of the antibodies describedherein, (e.g., any one of Hum231#1, Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15, orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161) and/or oneor more Chothia VH CDRs of a VH of any one of the antibodies describedherein (e.g., any one of antibodies Hum231#1, Hum231#2, pab1964,pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972,pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, orpab2161,). In certain embodiments, antibodies or fragments thereof thatspecifically bind to GITR (e.g., human GITR) comprise one or more CDRs,in which the Chothia and Kabat CDRs have the same amino acid sequence.In certain embodiments, provided herein are antibodies or fragmentsthereof that specifically bind to GITR (e.g., human GITR) and comprisecombinations of Kabat CDRs and Chothia CDRs. In a particular embodiment,provided herein are antibodies or fragments thereof that specificallybind to GITR (e.g., human GITR) and comprise Chothia CDRs of any of theantibodies described herein (e.g., Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, orpab2161).

In certain aspects, the CDRs of an antibody can be determined accordingto the IMGT numbering system as described in Lefranc M-P, (1999) TheImmunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is atpositions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is atpositions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is atpositions 50 to 52, and VL-CDR3 is at positions 89 to 97. In aparticular embodiment, provided herein are antibodies or fragmentsthereof that specifically bind to GITR (e.g., human GITR) and compriseCDRs of any one of the antibodies described herein (e.g., any one ofantibodies Hum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967,pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976,pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies1-107, or antibodies pab2159, pab2160, or pab2161), which are determinedby the IMGT numbering system, for example, as described in Lefranc M-P(1999) supra and Lefranc M-P et al., (1999) supra).

In certain aspects, the CDRs of an antibody can be determined accordingto MacCallum R M et al., (1996) J Mol Biol 262: 732-745. See also, e.g.,Martin A. “Protein Sequence and Structure Analysis of Antibody VariableDomains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter31, pp. 422-439, Springer-Verlag, Berlin (2001). In a particularembodiment, provided herein are antibodies or fragments thereof thatspecifically bind to GITR (e.g., human GITR) and comprise CDRs of anyone of the antibodies described herein (e.g., any one of antibodiesHum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161), which are determined by themethod in MacCallum R M et al.

In certain aspects, the CDRs of an antibody can be determined accordingto the AbM numbering scheme, which refers AbM hypervariable regionswhich represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software (Oxford Molecular Group, Inc.). In a particularembodiment, provided herein are antibodies or fragments thereof thatspecifically bind to GITR (e.g., human GITR) and comprise CDRs of anyone of the antibodies described herein (e.g., any one of antibodiesHum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161), which are determined by theAbM numbering scheme.

In a specific embodiment, the position of one or more CDRs along the VH(e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) regionof an antibody described herein may vary by one, two, three, four, five,or six amino acid positions so long as immunospecific binding to GITR(e.g., human GITR) is maintained (e.g., substantially maintained, forexample, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%). For example, in one embodiment, the positiondefining a CDR of any of antibody described herein (e.g., Hum231#1,Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, 231-32-15, or antibodies 1-107, or antibodies pab2159,pab2160, or pab2161) may vary by shifting the N-terminal and/orC-terminal boundary of the CDR by one, two, three, four, five, or sixamino acids, relative to the CDR position of any one of the antibodiesdescribed herein (e.g., Hum231#1, Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15, orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161, identifiedin, e.g., Table 1), so long as immunospecific binding to GITR (e.g.,human GITR) is maintained (e.g., substantially maintained, for example,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%). In another embodiment, the length of one or more CDRs alongthe VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3)region of an antibody described herein may vary (e.g., be shorter orlonger) by one, two, three, four, five, or more amino acids, so long asimmunospecific binding to GITR (e.g., human GITR) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%).

In one embodiment, a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/orVH CDR3 described herein may be one, two, three, four, five or moreamino acids shorter than one or more of the CDRs described herein (e.g.,SEQ ID NO: 1-34, 101-109, or 114-189 or SEQ ID NO: 35 or 191-194) solong as immunospecific binding to GITR (e.g., human GITR) is maintained(e.g., substantially maintained, for example, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%). In anotherembodiment, a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VHCDR3 described herein may be one, two, three, four, five or more aminoacids longer than one or more of the CDRs described herein (e.g., SEQ IDNO: 1-34, 101-109, or 114-189 or SEQ ID NO: 35 or 191-194) so long asimmunospecific binding to GITR (e.g., human GITR) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%). In anotherembodiment, the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1,VH CDR2, and/or VH CDR3 described herein may be extended by one, two,three, four, five or more amino acids compared to one or more of theCDRs described herein (e.g., SEQ ID NO: 1-34, 101-109, or 114-189 or SEQID NO: 35 or 191-194) so long as immunospecific binding to GITR (e.g.,human GITR) is maintained (e.g., substantially maintained, for example,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%). In another embodiment, the carboxy terminus of a VL CDR1, VLCDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein may beextended by one, two, three, four, five or more amino acids compared toone or more of the CDRs described herein (e.g., SEQ ID NO: 1-34,101-109, or 114-189 or SEQ ID NO: 35 or 191-194) so long asimmunospecific binding to GITR (e.g., human GITR) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%). In anotherembodiment, the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1,VH CDR2, and/or VH CDR3 described herein may be shortened by one, two,three, four, five or more amino acids compared to one or more of theCDRs described herein (e.g., SEQ ID NO: 1-34, 101-109, or 114-189 or SEQID NO: 35 or 191-194) so long as immunospecific binding to GITR (e.g.,human GITR) is maintained (e.g., substantially maintained, for example,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%). In one embodiment, the carboxy terminus of a VL CDR1, VLCDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein may beshortened by one, two, three, four, five or more amino acids compared toone or more of the CDRs described herein (e.g., SEQ ID NO: 1-34,101-109, or 114-189 or SEQ ID NO: 35 or 191-194) so long asimmunospecific binding to GITR (e.g., human GITR) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%). Any method knownin the art can be used to ascertain whether immunospecific binding toGITR (e.g., human GITR) is maintained, for example, the binding assaysand conditions described in the “Examples” section (Section 6) providedherein.

In specific aspects, provided herein is an antibody comprising anantibody light chain and heavy chain, e.g., a separate light chain andheavy chain. With respect to the light chain, in a specific embodiment,the light chain of an antibody described herein is a kappa light chain.In another specific embodiment, the light chain of an antibody describedherein is a lambda light chain. In yet another specific embodiment, thelight chain of an antibody described herein is a human kappa light chainor a human lambda light chain. In a particular embodiment, an antibodydescribed herein, which immunospecifically binds to an GITR polypeptide(e.g., human GITR) comprises a light chain wherein the amino acidsequence of the VL domain comprises any amino acid sequence describedherein (e.g., SEQ ID NO: 202, 204, 205, 207, 208, or 400-518), andwherein the constant region of the light chain comprises the amino acidsequence of a human kappa light chain constant region. In a particularembodiment, an antibody described herein, which immunospecifically bindsto an GITR polypeptide (e.g., human GITR) comprises a light chainwherein the amino acid sequence of the VL domain comprises any aminoacid sequence described herein (e.g., SEQ ID NO: 519), and wherein theconstant region of the light chain comprises the amino acid sequence ofa human kappa light chain constant region. In another particularembodiment, an antibody described herein, which immunospecifically bindsan GITR (e.g., human GITR) comprises a light chain wherein the aminoacid sequence of the VL domain can comprise any amino acid sequencedescribed herein (e.g., SEQ ID NO: 202, 204, 205, 207, 208, or 400-518),and wherein the constant region of the light chain comprises the aminoacid sequence of a human lambda light chain constant region. In anotherparticular embodiment, an antibody described herein, whichimmunospecifically binds an GITR (e.g., human GITR) comprises a lightchain wherein the amino acid sequence of the VL domain can comprise anyamino acid sequence described herein (e.g., SEQ ID NO: 519), and whereinthe constant region of the light chain comprises the amino acid sequenceof a human lambda light chain constant region. In a specific embodiment,an antibody described herein, which immunospecifically binds to GITR(e.g., human GITR) comprises a light chain wherein the amino acid of theVL domain comprises (SEQ ID NOs: 207 or 208) and wherein the constantregion of the light chain comprises the amino acid sequence of a humankappa or lambda light chain constant region. Non-limiting examples ofhuman constant region sequences have been described in the art, e.g.,see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra.

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR) comprises alight chain comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 555, 556, 571-576, and 580. In a particularembodiment, an antibody or fragment thereof described herein, whichspecifically binds to GITR (e.g., human GITR) comprises a light chaincomprising the amino acid sequence selected from the group consisting ofSEQ ID NOs: 571-576.

With respect to the heavy chain, in a specific embodiment, the heavychain of an antibody described herein can be an alpha (α), delta (δ),epsilon (ε), gamma (γ) or mu (μ) heavy chain. In another specificembodiment, the heavy chain of an antibody described can comprise ahuman alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavychain. In a particular embodiment, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a heavychain wherein the amino acid sequence of the VH domain can comprise anyamino acid sequence described herein (e.g., any of SEQ ID NO: 201, 203,206, or 215-389), and wherein the constant region of the heavy chaincomprises the amino acid sequence of a human gamma (γ) heavy chainconstant region. In a specific embodiment, an antibody described herein,which specifically binds to GITR (e.g., human GITR), comprises a heavychain wherein the amino acid sequence of the VH domain comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:553, 554, 567-570, and 579, and wherein the constant region of the heavychain comprises the amino acid of a human heavy chain described hereinor known in the art. In a specific embodiment, an antibody describedherein, which specifically binds to GITR (e.g., human GITR), comprises aheavy chain wherein the amino acid sequence of the VH domain comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:581 and 582, and wherein the constant region of the heavy chaincomprises the amino acid of a human heavy chain described herein orknown in the art. Non-limiting examples of human constant regionsequences have been described in the art, e.g., see U.S. Pat. No.5,693,780 and Kabat E A et al., (1991) supra.

In a particular embodiment, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprises aheavy chain comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 553, 554, 567-570, and 579. In a particularembodiment, an antibody or fragment thereof described herein, whichspecifically binds to GITR (e.g., human GITR), comprises a heavy chaincomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 581 and 582. In a specific embodiment, an antibody orfragment thereof, which binds to GITR (e.g., human GITR), comprises aheavy chain comprising the amino acid sequence of SEQ ID NOs: 567-570.

In a specific embodiment, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR) comprises a VLdomain and a VH domain comprising any amino acid sequences describedherein, and wherein the constant regions comprise the amino acidsequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgYimmunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA or IgYimmunoglobulin molecule. In another specific embodiment, an antibodydescribed herein, which immunospecifically binds to GITR (e.g., humanGITR) comprises a VL domain and a VH domain comprising any amino acidsequences described herein, and wherein the constant regions comprisethe amino acid sequences of the constant regions of an IgG, IgE, IgM,IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁ and IgA₂), or any subclass (e.g., IgG_(2a) andIgG_(2b)) of immunoglobulin molecule. In a particular embodiment, theconstant regions comprise the amino acid sequences of the constantregions of a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulinmolecule, any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂), orany subclass (e.g., IgG_(2a) and IgG_(2b)) of immunoglobulin molecule.

In another specific embodiment, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VLdomain and a VH domain comprising any amino acid sequences describedherein, and wherein the constant regions comprise the amino acidsequences of the constant regions of a human IgG₁ (e.g., allotypes G1m3,G1m17,1 or G1m17,1,2) or human IgG₄. In a particular embodiment, anantibody described herein, which immunospecifically binds to an GITR(e.g., human GITR) comprises a VL domain and a VH domain comprising anyamino acid sequences described herein, and wherein the constant regionscomprise the amino acid sequences of the constant region of a human IgG₁(allotype Glm3). Non-limiting examples of human constant regions aredescribed in the art, e.g., see Kabat E A et al., (1991) supra.

In another embodiment, an antibody or fragment thereof described herein,which specifically binds to GITR (e.g., human GITR) comprises a lightchain comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 555, 556, 571-576, and 580 and a heavy chaincomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 553, 554, 567-570, and 579. In another embodiment, anantibody or fragment thereof described herein, which specifically bindsto GITR (e.g., human GITR) comprises a light chain comprising the aminoacid sequence of SEQ ID NO:576 and a heavy chain comprising the aminoacid sequence selected from the group consisting of SEQ ID NO: 581 and582. In a specific embodiment, an antibody or fragment thereof, whichspecifically binds to GITR (e.g., human GITR), comprises a light chaincomprising the amino acid sequence of SEQ ID NO: 555 or 556 and a heavychain comprising the amino acid sequence of SEQ ID NO: 554.

In certain embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein or a fragment thereof (e.g., CH2 domain (residues231-340 of human IgG₁) and/or CH3 domain (residues 341-447 of humanIgG₁) and/or the hinge region, with numbering according to the Kabatnumbering system (e.g., the EU index in Kabat)) to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding and/or antigen-dependentcellular cytotoxicity.

In certain embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain may be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein or a fragment thereof (e.g., CH2 domain (residues231-340 of human IgG₁) and/or CH3 domain (residues 341-447 of humanIgG₁) and/or the hinge region, with numbering according to the Kabatnumbering system (e.g., the EU index in Kabat)) to increase or decreasethe affinity of the antibody for an Fc receptor (e.g., an activated Fcreceptor) on the surface of an effector cell. Mutations in the Fc regionof an antibody or fragment thereof that decrease or increase theaffinity of an antibody for an Fc receptor and techniques forintroducing such mutations into the Fc receptor or fragment thereof areknown to one of skill in the art. Examples of mutations in the Fcreceptor of an antibody that can be made to alter the affinity of theantibody for an Fc receptor are described in, e.g., Smith P et al.,(2012) PNAS 109: 6181-6186, U.S. Pat. No. 6,737,056, and InternationalPublication Nos. WO 02/060919; WO 98/23289; and WO 97/34631, which areincorporated herein by reference.

In a specific embodiment, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo. In some embodiments, one, two or more amino acidmutations (i.e., substitutions, insertions or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to decrease the half-lifeof the antibody in vivo. In other embodiments, one, two or more aminoacid mutations (i.e., substitutions, insertions or deletions) areintroduced into an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In a specific embodiment, the antibodies mayhave one or more amino acid mutations (e.g., substitutions) in thesecond constant (CH2) domain (residues 231-340 of human IgG₁) and/or thethird constant (CH3) domain (residues 341-447 of human IgG₁), withnumbering according to the EU index in Kabat (Kabat E A et al., (1991)supra). In a specific embodiment, the constant region of the IgG₁ of anantibody or antigen-binding fragment thereof described herein comprisesa methionine (M) to tyrosine (Y) substitution in position 252, a serine(S) to threonine (T) substitution in position 254, and a threonine (T)to glutamic acid (E) substitution in position 256, numbered according tothe EU index as in Kabat. See U.S. Pat. No. 7,658,921, which isincorporated herein by reference. This type of mutant IgG, referred toas “YTE mutant” has been shown to display fourfold increased half-lifeas compared to wild-type versions of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24). In certain embodiments, anantibody or antigen-binding fragment thereof comprises an IgG constantdomain comprising one, two, three or more amino acid substitutions ofamino acid residues at positions 251-257, 285-290, 308-314, 385-389, and428-436, numbered according to the EU index as in Kabat.

In a further embodiment, one, two or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322, numbered according to the EU index as in Kabat, can be replacedwith a different amino acid residue such that the antibody has analtered affinity for an effector ligand but retains the antigen-bindingability of the parent antibody. The effector ligand to which affinity isaltered can be, for example, an Fc receptor or the C1 component ofcomplement. This approach is described in further detail in U.S. Pat.Nos. 5,624,821 and 5,648,260. In some embodiments, the deletion orinactivation (through point mutations or other means) of a constantregion domain may reduce Fc receptor binding of the circulating antibodythereby increasing tumor localization. See, e.g., U.S. Pat. Nos.5,585,097 and 8,591,886 for a description of mutations that delete orinactivate the constant domain and thereby increase tumor localization.In certain embodiments, one or more amino acid substitutions may beintroduced into the Fc region of an antibody described herein to removepotential glycosylation sites on Fc region, which may reduce Fc receptorbinding (see, e.g., Shields R L et al., (2001) J Biol Chem 276:6591-604). In various embodiments, one or more of the followingmutations in the constant region of an antibody described herein may bemade: an N297A substitution; an N297Q substitution; a L235A substitutionand a L237A substitution; a L234A substitution and a L235A substitution;a E233P substitution; a L234V substitution; a L235A substitution; a C236deletion; a P238A substitution; a D265A substitution; a A327Qsubstitution; or a P329A substitution, numbered according to the EUindex as in Kabat.

In a specific embodiment, an antibody or antigen-binding fragmentthereof described herein comprises the constant domain of an IgG₁ withan N297Q or N297A amino acid substitution.

In certain embodiments, one or more amino acids selected from amino acidresidues 329, 331 and 322 in the constant region of an antibodydescribed herein, numbered according to the EU index as in Kabat, can bereplaced with a different amino acid residue such that the antibody hasaltered C1q binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al). In some embodiments, one or moreamino acid residues within amino acid positions 231 to 238 in theN-terminal region of the CH2 domain of an antibody described herein arealtered to thereby alter the ability of the antibody to fix complement.This approach is described further in International Publication No. WO94/29351. In certain embodiments, the Fc region of an antibody describedherein is modified to increase the ability of the antibody to mediateantibody dependent cellular cytotoxicity (ADCC) and/or to increase theaffinity of the antibody for an Fcγ receptor by mutating one or moreamino acids (e.g., introducing amino acid substitutions) at thefollowing positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265,267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292,293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322,324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373,376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439, numbered according to the EU index as in Kabat. This approach isdescribed further in International Publication No. WO 00/42072.

In certain embodiments, an antibody described herein comprises theconstant region of an IgG₄ antibody and the serine at amino acid residue228 of the heavy chain, numbered according to the EU index as in Kabat,is substituted for proline.

Antibodies with reduced fucose content have been reported to have anincreased affinity for Fc receptors, such as, e.g., FcγRIIIa.Accordingly, in certain embodiments, the antibodies or antigen-bindingfragments thereof described herein have reduced fucose content or nofucose content. Such antibodies can be produced using techniques knownto one skilled in the art. For example, the antibodies can be expressedin cells deficient or lacking the ability of fucosylation. In a specificexample, cell lines with a knockout of both alleles ofα1,6-fucosyltransferase can be used to produce antibodies with reducedfucose content. The Potelligent® system (Lonza) is an example of such asystem that can be used to produce antibodies with reduced fucosecontent. Alternatively, antibodies or antigen-binding fragments withreduced fucose content or no fucose content can be produced by, e.g.:(i) culturing cells under conditions which prevent or reducefucosylation; (ii) posttranslational removal of fucose (e.g., with afucosidase enzyme); (iii) post-translational addition of the desiredcarbohydrate, e.g., after recombinant expression of a non-glycosylatedglycoprotein; or (iv) purification of the glycoprotein so as to selectfor antibodies or antigen-binding fragments thereof which are notfucosylated. See, e.g., Longmore G D & Schachter H (1982) Carbohydr Res100: 365-92 and Imai-Nishiya H et al., (2007) BMC Biotechnol. 7: 84 formethods for producing antibodies or antigen-binding fragments thereofwith no fucose content or reduced fucose content.

In certain embodiments, antibodies or antigen-binding fragments thereofdescribed herein have an increased affinity for CD32B (also known asFcγRIIB or FCGR2B), e.g., as compared to an antibody with a wild-type Fcregion, e.g., an IgG1 Fc. In certain embodiments, antibodies orantigen-binding fragments thereof described herein have a selectivelyincreased affinity for CD32B (FcγRIIB) over both CD32A (FcγRIIA) andCD16 (FcγRIIIA). Sequence alterations that result in increased affinityfor CD32B are provided, for example, in Mimoto et al., ProteinEngineering, Design & Selection 10: 589-598 (2013), Chu et al.,Molecular Immunology 45: 3926-3933 (2008), and Strohl, Current Opinionin Biology 20: 685-691 (2009), each of which is herein incorporated byreference in its entirety. In some embodiments, the antibody orantigen-binding fragment with an increased affinity for CD32B comprisesa heavy chain constant region, e.g., an IgG1 constant region, orfragment thereof comprising a mutation selected from the groupconsisting of: G236D, P238D, S239D, S267E, L328F, L328E, an arginineinserted after position 236, and combinations thereof, numberedaccording to EU index (Kabat et al., Sequences of Proteins ofImmunological Interest, U.S. Department of Health and Human Services,Bethesda (1991)). In some embodiments, the antibody or antigen-bindingfragment with an increased affinity for CD32B comprises a heavy chainconstant region, e.g., an IgG1 constant region, or fragment thereofcomprising S267E and L328F substitutions. In some embodiments, theantibody or antigen-binding fragment with an increased affinity forCD32B comprises a heavy chain constant region, e.g., an IgG1 constantregion, or fragment thereof comprising P238D and L328E substitutions. Insome embodiments, the antibody or antigen-binding fragment with anincreased affinity for CD32B comprises a heavy chain constant region,e.g., an IgG1 constant region, or fragment thereof comprising a P238Dsubstitution and substitution selected from the group consisting ofE233D, G237D, H268D, P271G, A330R, and combinations thereof. In someembodiments, the antibody or antigen-binding fragment with an increasedaffinity for CD32B comprises a heavy chain constant region, e.g., anIgG1 constant region, or fragment thereof comprising P238D, E233D,G237D, H268D, P271G, and A330R substitutions. In some embodiments, theantibody or antigen-binding fragment with an increased affinity forCD32B comprises a heavy chain constant region, e.g., an IgG1 constantregion, or fragment thereof comprising G236D and S267E. In someembodiments, the antibody or antigen-binding fragment with an increasedaffinity for CD32B comprises a heavy chain constant region, e.g., anIgG1 constant region, or fragment thereof comprising S239D and S267E. Insome embodiments, the antibody or antigen-binding fragment with anincreased affinity for CD32B comprises a heavy chain constant region,e.g., an IgG1 constant region, or fragment thereof comprising S267E andL328F. In some embodiments, the antibody or antigen-binding fragmentwith an increased affinity for CD32B comprises a heavy chain constantregion, e.g., an IgG1 constant region, or fragment thereof comprising anarginine inserted after position 236 and L328R.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a lightchain and a heavy chain, wherein (i) the light chain comprises a VLdomain comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequences of any one of Hum231#2, pab1964, pab1965, pab1966, pab1967,pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976,pab1977, pab1979, pab1980, pab1981, pab1983, Hum231#1, 231-32-15, orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161 (e.g.,those listed in Table 1); (ii) the heavy chain comprises a VH domaincomprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acidsequences of any one of Hum231#2, pab1964, pab1965, pab1966, pab1967,pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976,pab1977, pab1979, pab1980, pab1981, pab1983, Hum231#1, 231-32-15, orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161 (e.g.,those listed in Table 2); (iii) the light chain further comprises aconstant light chain domain comprising the amino acid sequence of theconstant domain of a human kappa light chain; and (iv) the heavy chainfurther comprises a constant heavy chain domain comprising the aminoacid sequence of the constant domain of a human IgG₁ (optionally IgG₁(allotype Glm3)) heavy chain.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a lightchain and a heavy chain, wherein (i) the light chain comprises a VLdomain comprising the amino acid sequence of any one of the antibodiesHum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, Hum231#1, 231-32-15, or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161, (e.g., SEQ ID NO: 202, 204,205, 207, 208, or 400-518 or SEQ ID NO:519); (ii) the heavy chaincomprises a VH domain comprising the amino acid sequence of and one ofthe antibodies Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, Hum231#1, 231-32-15, or antibodies1-107, or antibodies pab2159, pab2160, or pab2161, (e.g., SEQ ID NO:201, 203, 206, or 215-389); (iii) the light chain further comprises aconstant domain comprising the amino acid sequence of the constantdomain of a human kappa light chain; and (iv) the heavy chain furthercomprises a constant domain comprising the amino acid sequence of theconstant domain of a human IgG₁ (optionally IgG₁ (allotype Glm3)) heavychain.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a lightchain and a heavy chain, wherein (i) the light chain comprises a VLdomain comprising the amino acid sequence of Hum231#1 or Hum231#2 (e.g.,SEQ ID NO: 207 or 208); (ii) the heavy chain comprises a VH domaincomprising the amino acid sequence of Hum231#1 or Hum231#2 (e.g., SEQ IDNO: 206); (iii) the light chain further comprises a constant domaincomprising the amino acid sequence of the constant domain of a humankappa light chain; and (iv) the heavy chain further comprises a constantdomain comprising the amino acid sequence of the constant domain of ahuman IgG₁ (optionally IgG₁ (allotype Glm3)) heavy chain.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a lightchain and a heavy chain, wherein (i) the light chain comprises a VLdomain comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequences of any one of the antibodies described herein, e.g., Hum231#2,pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971,pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981,pab1983, Hum231#1, 231-32-15, or antibodies 1-107, or antibodiespab2159, pab2160, or pab2161 (e.g., those listed in Table 1); (ii) theheavy chain comprises a VH domain comprising a VH CDR1, VH CDR2, and VHCDR3 having the amino acid sequences of any one of the antibodiesdescribed herein, e.g., Hum231#2, pab1964, pab1965, pab1966, pab1967,pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976,pab1977, pab1979, pab1980, pab1981, pab1983, Hum231#1, 231-32-15, orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161 (e.g.,those listed in Table 2); (iii) the light chain further comprises aconstant light chain domain comprising the amino acid sequence of theconstant domain of a human IgG₄; and (iv) the heavy chain furthercomprises a constant heavy chain domain comprising the amino acidsequence of the constant domain of a human IgG₄ heavy chain.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a lightchain and a heavy chain, wherein (i) the light chain comprises a VLdomain comprising the amino acid sequence of any one of the antibodiesdescribed herein, e.g., Hum231#2, pab1964, pab1965, pab1966, pab1967,pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976,pab1977, pab1979, pab1980, pab1981, pab1983, Hum231#1, 231-32-15, orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161 (e.g., SEQID NO: 202, 204, 205, 207, 208, or 400-518 or SEQ ID NO: 519); (ii) theheavy chain comprises a VH domain comprising the amino acid sequence ofany one of the antibodies described herein, e.g., Hum231#2, pab1964,pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972,pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,Hum231#1, 231-32-15, or antibodies 1-107, or antibodies pab2159,pab2160, or pab2161 (e.g., SEQ ID NO: 201, 203, 206, or 215-389); (iii)the light chain further comprises a constant domain comprising the aminoacid sequence of the constant domain of a human IgG₄ light chain; and(iv) the heavy chain further comprises a constant domain comprising theamino acid sequence of the constant domain of a human IgG₄ heavy chain.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a lightchain and a heavy chain, wherein (i) the light chain comprises a VLdomain comprising the amino acid sequence of either Hum231#1 or Hum231#2(e.g., SEQ ID NO: 207 or 208); (ii) the heavy chain comprises a VHdomain comprising the amino acid sequence of either Hum231#1 or Hum231#2(e.g., SEQ ID NO: 206); (iii) the light chain further comprises aconstant domain comprising the amino acid sequence of the constantdomain of a human IgG₄ light chain; and (iv) the heavy chain furthercomprises a constant domain comprising the amino acid sequence of theconstant domain of a human IgG₄ heavy chain.

In a specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 553, 554, and 567 to 570; and (b) a light chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 555, 556, and 571 to 576. In a specific embodiment, anantibody provided herein, which specifically binds to GITR (e.g., humanGITR), comprises (a) a heavy chain comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 581 or 582; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 576. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 553; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 556. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 554; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 556. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 581; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 556. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 582; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 556. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 553; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 555. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 554; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 555. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 567; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 573. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 567; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 576. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 554; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 576. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 581; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 576. Inanother specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 582; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 576.

In another specific embodiment, an antibody provided herein, whichspecifically binds to GITR (e.g., human GITR), comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 553 with an aminoacid substitution of N to A or Q at amino acid position 298; and (b) alight chain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 555, 556, and 571 to 576. In another specificembodiment, an antibody provided herein, which specifically binds toGITR (e.g., human GITR), comprises (a) a heavy chain comprising theamino acid sequence of SEQ ID NO: 553 with an amino acid substitution ofN to A or Q at amino acid position 298; and (b) a light chain comprisingthe amino acid sequence of SEQ ID NO: 556. In another specificembodiment, an antibody provided herein, which specifically binds toGITR (e.g., human GITR), comprises (a) a heavy chain comprising theamino acid sequence of SEQ ID NO: 553 with an amino acid substitution ofN to A or Q at amino acid position 298; and (b) a light chain comprisingthe amino acid sequence of SEQ ID NO: 555.

In certain embodiments, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesone, two, three, or four VL framework regions (FRs) having the aminoacid sequence described herein for any one of the antibodies describedherein, e.g., Hum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967,pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976,pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15, or antibodies1-107, or antibodies pab2159, pab2160, or pab2161 (e.g., see Table 3).In some embodiments an antibody or fragment thereof, which specificallybinds to GITR (e.g., human GITR) comprises one, two, three, or four VHframework regions (FRs) having the amino acid sequence described hereinfor any one of the antibodies described herein, e.g., Hum231#1,Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, 231-32-15, or antibodies 1-107, or antibodies pab2159,pab2160, or pab2161 (e.g., see Table 4). In certain embodiments, anantibody or fragment thereof, which specifically binds to GITR (e.g.,human GITR), comprises one, two, three, four, five, six, seven, or eightof the FRs of one of the antibodies described herein (e.g., Hum231#1Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, 231-32-15, or antibodies 1-107, or antibodies pab2159,pab2160, or pab2161). In specific embodiments, an antibody or fragmentthereof described herein, which immunospecifically binds to GITR (e.g.,human GITR), comprises framework regions (e.g., framework regions of theVL domain and/or VH domain) that are human framework regions or derivedfrom human framework regions. Non-limiting examples of human frameworkregions are described in the art, e.g., see Kabat E A et al., (1991)supra). In certain embodiment, an antibody described herein comprisesframework regions (e.g., framework regions of the VL domain and/or VHdomain) that are primate (e.g., non-human primate) framework regions orderived from primate (e.g., non-human primate) framework regions.

For example, CDRs from antigen-specific non-human antibodies, typicallyof rodent origin (e.g., mouse or rat), are grafted onto homologous humanor non-human primate acceptor frameworks. In one embodiment, thenon-human primate acceptor frameworks are from Old World apes. In aspecific embodiment, the Old World ape acceptor framework is from Pantroglodytes, Pan paniscus or Gorilla gorilla. In a particularembodiment, the non-human primate acceptor frameworks are from thechimpanzee Pan troglodytes. In a particular embodiment, the non-humanprimate acceptor frameworks are Old World monkey acceptor frameworks. Ina specific embodiment, the Old World monkey acceptor frameworks are fromthe genus Macaca. In a certain embodiment, the non-human primateacceptor frameworks are is derived from the cynomolgus monkey Macacacynomolgus. Non-human primate framework sequences are described in U.S.Patent Application Publication No. US 2005/0208625.

In certain embodiments, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesone, two or more VL framework regions (FRs) having the amino acidsequences described herein for any one of the antibodies set forth inTable 3, supra. In some embodiments, an antibody or fragment thereofdescribed herein, which specifically binds to GITR (e.g., human GITR),comprises one, two or more VH framework regions (FRs) having the aminoacid sequences described herein for any one of the antibodies set forthin Table 4, supra. In specific embodiments, an antibody or fragmentthereof described herein, which specifically binds to GITR (e.g., humanGITR), comprises one, two or more VL framework regions having the aminoacid sequences described herein for any one of the antibodies set forthin Table 3, supra, and one, two or more VH framework regions having theamino acid sequences described herein for the antibodies set forth inTable 4, supra.

In certain embodiments, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesthe framework regions of VL domain having the amino acid sequenceselected from the group consisting of SEQ ID NO: 202, 207, 208, 400-411,413-416, 418-421, 423-448, 450-452, 454-464, 467-477, 481-486, 488-513,or 515-518 and/or the framework regions of the VH domain having theamino acid sequence selected from the group consisting of SEQ ID NO:201, 206, 215, 217-234, 236-256, 258, 259, 261-265, 267, 268, 271-273,276, 277, 280, 281, 283-285, 287, 288, 290, 291, 294, 296-299, 301,304-306, 308, 313-316, 319, 320, 322-325, 327, 328, 333, 336, 338-340,342, 343, 345, 350, 354-356, 358-360, 362-368, 380, 384, or 387. Incertain embodiments, an antibody or fragment thereof described herein,which specifically binds to GITR (e.g., human GITR), comprises theframework regions of VL domain having the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 202, 207, 208, 400-411, 413-416,418-421, 423-448, 450-464, 467-477, 481-486, 488-513, or 515-519 and/orthe framework regions of the VH domain having the amino acid sequenceselected from the group consisting of SEQ ID NO: 201, 206, 215, 217-234,236-256, 258, 259, 261-265, 267, 268, 270-273, 276, 277, 280, 281,283-285, 287, 288, 290, 291, 294, 296-299, 301, 304-306, 308, 313-316,319, 320, 322-325, 327, 328, 333, 336, 338-340, 342, 343, 345, 350,354-356, 358-360, or 362-368. In some embodiments, an antibody orfragment thereof described herein, which specifically binds to GITR(e.g., human GITR), comprises one, two, three or four framework regionsof the VL domain having the amino acid sequence of any one of theantibodies described herein, e.g., Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107 (e.g., SEQ ID NO: 202, 207, 208, 400-411,413-416, 418-421, 423-448, 450-452, 454-464, 467-477, 481-486, 488-513,or 515-518) with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid mutations(e.g., amino acid substitutions, such as conservative amino acidsubstitutions) and/or the framework regions of the VH domain having theamino acid sequence of any one of the antibodies described herein, e.g.,Hum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15, or antibodies 1-107(e.g., SEQ ID NO: 201, 206, 215, 217-234, 236-256, 258, 259, 261-265,267, 268, 271-273, 276, 277, 280, 281, 283-285, 287, 288, 290, 291, 294,296-299, 301, 304-306, 308, 313-316, 319, 320, 322-325, 327, 328, 333,336, 338-340, 342, 343, 345, 350, 354-356, 358-360, 362-368, 380, 384,or 387). In some embodiments, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesone, two, three or four framework regions of the VL domain having theamino acid sequence of any one of the antibodies described herein, e.g.,Hum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15, or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161 (e.g., SEQ ID NO: 202, 207, 208,400-411, 413-416, 418-421, 423-448, 450-464, 467-477, 481-486, 488-513,or 515-519) with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid mutations(e.g., amino acid substitutions, such as conservative amino acidsubstitutions) and/or the framework regions of the VH domain having theamino acid sequence of any one of the antibodies described herein, e.g.,Hum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15, or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161 (e.g., SEQ ID NO: 201, 206, 215,217-234, 236-256, 258, 259, 261-265, 267, 268, 270-273, 276, 277, 280,281, 283-285, 287, 288, 290, 291, 294, 296-299, 301, 304-306, 308,313-316, 319, 320, 322-325, 327, 328, 333, 336, 338-340, 342, 343, 345,350, 354-356, 358-360, or 362-368). In certain embodiments, an antibodyor fragment thereof described herein, which specifically binds to GITR(e.g., human GITR), comprises one, two, three or four framework regionsof the VH domain having the amino acid sequence of any one of theantibodies described herein, e.g., Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, pab2161(e.g., SEQ ID NO: 201, 203, 206, or 215-389) with 1, 2, 3, 4, 5, 6, 7,8, 9 or more amino acid mutations (e.g., amino acid substitutions, suchas conservative amino acid substitutions) and/or the framework regionsof the VL domain having the amino acid sequence of any one of theantibodies described herein, e.g., Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, orpab2161 (e.g., SEQ ID NO: 201, 203, 206, or 215-389). In someembodiments, an antibody or fragment thereof described herein, whichspecifically binds to GITR (e.g., human GITR), comprises one, two, threeor four framework regions of the VL domain having the amino acidsequence of any one of the antibodies described herein, e.g., Hum231#1,Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, 231-32-15, or antibodies 1-107 (e.g., SEQ ID NO: 202,207, 208, 400-411, 413-416, 418-421, 423-448, 450-452, 454-464, 467-477,481-486, 488-513, or 515-518) with 1, 2, 3, 4, 5, 6, 7, 8, 9 or moreamino acid mutations (e.g., amino acid substitutions, such asconservative amino acid substitutions) and/or one, two, three or fourframework regions of the VH domain having the amino acid sequence of SEQID NO: 201, 203, 206, or 215-389 with 1, 2, 3, 4, 5, 6, 7, 8, 9 or moreamino acid mutations (e.g., amino acid substitutions, such asconservative amino acid substitutions). In some embodiments, an antibodyor fragment thereof described herein, which specifically binds to GITR(e.g., human GITR), comprises one, two, three or four framework regionsof the VL domain having the amino acid sequence of any one of theantibodies described herein, e.g., Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, orpab2161 (e.g., SEQ ID NO: 202, 207, 208, 400-411, 413-416, 418-421,423-448, 450-464, 467-477, 481-486, 488-513, or 515-519) with 1, 2, 3,4, 5, 6, 7, 8, 9 or more amino acid mutations (e.g., amino acidsubstitutions, such as conservative amino acid substitutions) and/orone, two, three or four framework regions of the VH domain having theamino acid sequence of SEQ ID NO: 201, 203, 206, or 215-389 with 1, 2,3, 4, 5, 6, 7, 8, 9 or more amino acid mutations (e.g., amino acidsubstitutions, such as conservative amino acid substitutions).

In certain embodiments, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesVL framework regions (FRs) having at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the VL frameworkregions described herein in Table 3, supra. In certain embodiments, anantibody or fragment thereof described herein, which specifically bindsto GITR (e.g., human GITR), comprises VH framework regions (FRs) havingat least 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the VH framework regions described herein Table 4,supra. In some embodiments, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesVH framework regions (FRs) having at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the VH frameworkregions described herein Table 4, supra, and VL framework regions (FRs)having at least 80%, at least 85%, at least 90%, at least 95%, or atleast 98% sequence identity to the VL framework regions described hereinTable 3, supra.

In certain embodiments, an antibody or fragment thereof describedherein, which specifically binds to GITR (e.g., human GITR), comprisesVL framework regions (FRs) having at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the VL frameworkregions described herein for antibody Hum231#1, Hum231#2, pab1964,pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972,pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, orpab2161 (e.g., as set forth in Table 3). In some embodiments, anantibody or fragment thereof described herein, which specifically bindsto GITR (e.g., human GITR), comprises VH framework regions (FRs) havingat least 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the VH framework regions described herein forantibody Hum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967,pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976,pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15, or antibodies1-107, or antibodies pab2159, pab2160, or pab2161 (e.g., as set forth inTable 4). In certain embodiments, an antibody or fragment thereofdescribed herein, which specifically binds to GITR (e.g., human GITR),comprises: (i) VL framework regions (FRs) having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe VL framework regions described herein for Hum231#1, Hum231#2,pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971,pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981,pab1983, 231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160,or pab2161 (e.g., as set forth in Table 3); and (ii) VH frameworkregions (FRs) having at least 80%, at least 85%, at least 90%, at least95%, or at least 98% sequence identity to the VH framework regionsdescribed herein for Hum231#1, Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15, orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161 (e.g., asset forth in Table 4).

The determination of percent identity between two sequences (e.g., aminoacid sequences or nucleic acid sequences) can also be accomplished usinga mathematical algorithm. A specific, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin S & Altschul S F (1990) PNAS 87: 2264-2268,modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877. Suchan algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul S F et al., (1990) J Mol Biol 215: 403. BLAST nucleotidesearches can be performed with the NBLAST nucleotide program parametersset, e.g., for score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules described herein. BLAST proteinsearches can be performed with the XBLAST program parameters set, e.g.,to score 50, wordlength=3 to obtain amino acid sequences homologous to aprotein molecule described herein. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul S F et al., (1997) Nuc Acids Res 25: 3389 3402. Alternatively,PSI BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,National Center for Biotechnology Information (NCBI) on the worldwideweb, ncbi.nlm.nih.gov). Another specific, non limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VLdomain having at least 80%, at least 85%, at least 90%, at least 95%, orat least 98% sequence identity to the amino acid sequence of the VLdomain of any one of antibodies Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, Hum231#1,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, orpab2161 (e.g., SEQ ID NO: 202, 204, 205, 207, 208, or 400-518 or SEQ IDNO:519). In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VLdomain having at least 80%, at least 85%, at least 90%, at least 95%, orat least 98% sequence identity to the amino acid sequence of the VLdomain of any one of antibodies Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, Hum231#1,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, orpab2161, (e.g., SEQ ID NO: 202, 204, 205, 207, 208, or 400-518 or SEQ IDNO: 519), wherein the antibody or antigen-binding fragment comprisesCDRs (e.g., VL CDRs) that are identical to the CDRs (e.g., VL CDRs) ofan antibody set forth in Table 1 and/or Table 2 (e.g., the CDRs areidentical to the CDRs of a particular antibody referred to by name inTables 1 and/or 2).

In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VLdomain comprising VL framework regions having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of the framework regions selected from the groupconsisting of SEQ ID NO: 202, 204, 205, 207, 208, and 400-518. Incertain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VLdomain comprising VL framework regions having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of SEQ ID NO: 519. In a particular embodiment,the antibody comprises VL CDRs that are identical to the VL CDRs of anantibody set forth in Table 1 (e.g., the VL CDRs in one row in Table 1).

In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VHdomain having at least 80%, at least 85%, at least 90%, at least 95%, orat least 98% sequence identity to the amino acid sequence of the VHdomain of SEQ ID NO: 201, 203, 206, or 215-389. In certain embodiments,an antibody or fragment thereof, which immunospecifically binds to GITR(e.g., human GITR), comprises a VH domain having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of the VH domain of SEQ ID NO: 201, 203, 206, or215-389, wherein the antibody or antigen-binding fragment comprises CDRs(e.g., VL CDRs) that are identical to the CDRs (e.g., VL CDRs) of anantibody set forth in Table 1 and/or Table 2 (e.g., the CDRs areidentical to the CDRs of a particular named antibody referred to by namein Tables 1 and/or 2).

In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VHdomain comprising VH framework regions having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of the framework regions selected from the groupconsisting of SEQ ID NO: 201, 203, 206, and 215-389. In a particularembodiment, the antibody comprises VH CDRs that are identical to the VHCDRs of an antibody set forth in Table 2 (e.g., the VH CDRs in one rowin Table 2).

In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises: (i) a VLdomain having at least 80%, at least 85%, at least 90%, at least 95%, orat least 98% sequence identity to the amino acid sequence of the VLdomain selected from the group consisting of SEQ ID NO: 202, 204, 205,207, 208, and 400-518; and (ii) a VL domain having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% sequence identityto the amino acid sequence of the VH domain of SEQ ID NO: 201, 203, 206,or 215-389. In certain embodiments, an antibody or fragment thereof,which immunospecifically binds to GITR (e.g., human GITR), comprises:(i) a VL domain having at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 519; and (ii) a VL domain having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of the VH domain of SEQ ID NO: 304. In certainembodiments, an antibody or fragment thereof, which immunospecificallybinds to GITR (e.g., human GITR), comprises: (i) a VL domain having atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence of the VL domain selectedfrom the group consisting of SEQ ID NO: 202, 204, 205, 207, 208, and400-518; and (ii) a VH domain having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% sequence identity to the aminoacid sequence of the VH domain selected from the group of SEQ ID NO:201, 203, 206, and 215-389, wherein the antibody or antigen-bindingfragment comprises CDRs (e.g., VL CDRs) that are identical to the CDRs(e.g., VL CDRs) of an antibody set forth in Table 1 and/or Table 2(e.g., the CDRs are identical to the CDRs of a particular antibodyreferred to by name in Tables 1 and/or 2). In certain embodiments, anantibody or fragment thereof, which immunospecifically binds to GITR(e.g., human GITR), comprises: (i) a VL domain having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% sequence identityto the amino acid sequence of SEQ ID NO:519; and (ii) a VH domain havingat least 80%, at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence of SEQ ID NO:304, whereinthe antibody or antigen-binding fragment comprises CDRs (e.g., VL CDRs)that are identical to the CDRs (e.g., VL CDRs) of an antibody set forthin Table 1 and/or Table 2 (e.g., the CDRs are identical to the CDRs of aparticular antibody referred to by name in Tables 1 and/or 2).

In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises: (i) a VLdomain comprising VL framework regions having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of the framework regions selected from the groupconsisting of SEQ ID NO: 202, 204, 205, 207, and 208; and (ii) a VHdomain comprising VH framework regions having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of the framework regions selected from the groupconsisting of SEQ ID NO: 201, 203, 206, and 215-389. In a particularembodiment, the antibody comprises VL CDRs that are identical to the VLCDRs of an antibody set forth in Table 3 and/or VH CDRs that areidentical to the VH CDRs of an antibody set forth in Table 4.

In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VHdomain having at least 80%, at least 85%, at least 90%, at least 95%, orat least 98% sequence identity to the amino acid sequence of the VHdomain selected from the group of SEQ ID NO: 201, 203, 206, and 215-389.In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VHdomain having at least 80%, at least 85%, at least 90%, at least 95%, orat least 98% sequence identity to the amino acid sequence of the VHdomain selected from the group of SEQ ID NO: 201, 203, 206, and 215-389,wherein the antibody or antigen-binding fragment comprises CDRs (e.g.,VL CDRs) that are identical to the CDRs (e.g., VL CDRs) of an antibodyset forth in Table 1 and/or Table 2 (e.g., the CDRs are identical to theCDRs of a particular antibody referred to in Tables 1 and/or 2).

In certain embodiments, an antibody or fragment thereof, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VHdomain comprising VH framework regions having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of the framework regions selected from the groupconsisting of SEQ ID NO: 201, 203, 206, and 215-389. In a particularembodiment, the antibody comprises VH CDRs that are identical to the VHCDRs of an antibody set forth in Table 2 (e.g., the VH CDRs in one rowin Table 2).

In another aspect, provided herein are antibodies that bind the same oran overlapping epitope of GITR (e.g., an epitope of human GITR) as anantibody described herein (e.g., antibody Hum231#1, Hum231#2, pab1964,pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972,pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107), or antibodies pab2159, pab2160,pab2161, or Hum231#2w. In certain embodiments, the epitope of anantibody can be determined by, e.g., NMR spectroscopy, X-ray diffractioncrystallography studies, ELISA assays, hydrogen/deuterium exchangecoupled with mass spectrometry (e.g., liquid chromatography electrospraymass spectrometry), array-based oligo-peptide scanning assays, and/ormutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-raycrystallography, crystallization may be accomplished using any of theknown methods in the art (e.g., Giegé R et al., (1994) Acta CrystallogrD Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem189: 1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976)J Biol Chem 251: 6300-6303). Antibody:antigen crystals may be studiedusing well known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see e.g. Meth Enzymol (1985) volumes 114 &115, eds Wyckoff H W et al.; U.S. Patent Application No. 2004/0014194),and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter C W;Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies may be accomplished using anymethod known to one of skill in the art. See, e.g., Champe M et al.,(1995) supra and Cunningham B C & Wells J A (1989) supra for adescription of mutagenesis techniques, including alanine scanningmutagenesis techniques. In a specific embodiment, the epitope of anantibody or antigen-binding fragment thereof is determined using alaninescanning mutagenesis studies, such as described in Section 6, infra. Inaddition, antibodies that recognize and bind to the same or overlappingepitopes of GITR (e.g., human GITR) can be identified using routinetechniques such as an immunoassay, for example, by showing the abilityof one antibody to block the binding of another antibody to a targetantigen, i.e., a competitive binding assay. Competition binding assaysalso can be used to determine whether two antibodies have similarbinding specificity for an epitope. Competitive binding can bedetermined in an assay in which the immunoglobulin under test inhibitsspecific binding of a reference antibody to a common antigen, such asGITR. Numerous types of competitive binding assays are known, forexample: solid phase direct or indirect radioimmunoassay (RIA), solidphase direct or indirect enzyme immunoassay (EIA), sandwich competitionassay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253); solidphase direct biotin-avidin EIA (see Kirkland T N et al., (1986) JImmunol 137: 3614-9); solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see Harlow E & Lane D, (1988) Antibodies:A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using 1-125 label (see Morel G A et al., (1988) Mol Immunol 25(1):7-15); solid phase direct biotin-avidin EIA (Cheung R C et al., (1990)Virology 176: 546-52); and direct labeled RIA. (Moldenhauer G et al.,(1990) Scand J Immunol 32: 77-82). Typically, such an assay involves theuse of purified antigen (e.g., GITR such as human GITR) bound to a solidsurface or cells bearing either of these, an unlabeled testimmunoglobulin and a labeled reference immunoglobulin. Competitiveinhibition can be measured by determining the amount of label bound tothe solid surface or cells in the presence of the test immunoglobulin.Usually the test immunoglobulin is present in excess. Usually, when acompeting antibody is present in excess, it will inhibit specificbinding of a reference antibody to a common antigen by at least 50-55%,55-60%, 60-65%, 65-70% 70-75% or more. A competition binding assay canbe configured in a large number of different formats using eitherlabeled antigen or labeled antibody. In a common version of this assay,the antigen is immobilized on a 96-well plate. The ability of unlabeledantibodies to block the binding of labeled antibodies to the antigen isthen measured using radioactive or enzyme labels. For further detailssee, for example, Wagener C et al., (1983) J Immunol 130: 2308-2315;Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M et al.,(1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407 andAntibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, pp.386-389.

In one embodiment, a competition assay is performed using surfaceplasmon resonance (BIAcore®), e.g., by an ‘in tandem approach’ such asthat described by Abdiche Y N et al., (2009) Analytical Biochem 386:172-180, whereby GITR antigen is immobilized on the chip surface, forexample, a CM5 sensor chip and the anti-GITR antibodies are then runover the chip. To determine if an antibody competes with an anti-GITRantibody or antigen-binding fragment thereof described herein, theanti-GITR antibody is first run over the chip surface to achievesaturation and then the potential, competing antibody is added. Bindingof the competing antibody can then be determined and quantified relativeto a non-competing control.

In certain aspects, competition binding assays can be used to determinewhether an antibody is competitively blocked, e.g., in a dose dependentmanner, by another antibody for example, an antibody binds essentiallythe same epitope, or overlapping epitopes, as a reference antibody, whenthe two antibodies recognize identical or sterically overlappingepitopes in competition binding assays such as competition ELISA assays,which can be configured in all number of different formats, using eitherlabeled antigen or labeled antibody. In a particular embodiment, anantibody can be tested in competition binding assays with an antibodydescribed herein (e.g., antibody Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15or antibodies 1-107, or antibodies pab2159, pab2160, pab2161, orHum231#2w), or a chimeric or Fab antibody thereof, or an antibodycomprising VH CDRs and VL CDRs of an antibody described herein (e.g.,Hum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies 1-107, orantibodies pab2159, pab2160, pab2161, or Hum231#2w).

In another aspect, provided herein are antibodies that compete (e.g., ina dose dependent manner) for binding to GITR (e.g., human GITR) with anantibody described herein (e.g., Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15or antibodies 1-107), or antibodies pab2159, pab2160, pab2161, orHum231#2w, as determined using assays known to one of skill in the artor described herein (e.g., ELISA competitive assays or surface plasmonresonance). In another aspect, provided herein are antibodies thatcompetitively inhibit (e.g., in a dose dependent manner) an antibodydescribed herein (e.g., Hum231#1, Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15 orantibodies 1-107, or antibodies pab2159, pab2160, pab2161, or Hum231#2w)from binding to GITR (e.g., human GITR), as determined using assaysknown to one of skill in the art or described herein (e.g., ELISAcompetitive assays, or suspension array or surface plasmon resonanceassay described in Example 6, infra). In particular embodiments, suchcompetitively blocking antibody activates, induces or enhances one ormore GITR activities. In specific aspects, provided herein is anantibody which competes (e.g., in a dose dependent manner) for specificbinding to GITR (e.g., human GITR), with an antibody comprising theamino acid sequences described herein (e.g., VL and/or VH amino acidsequences of antibody Hum231#1, Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15 orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161, orHum231#2w), as determined using assays known to one of skill in the artor described herein (e.g., ELISA competitive assays, or suspension arrayor surface plasmon resonance assay described in Example 6, infra).

In certain embodiments, provided herein is an antibody that competeswith an antibody described herein for binding to GITR (e.g., human GITR)to the same extent that the antibody described herein self-competes forbinding to GITR (e.g., human GITR). In some embodiments, provided hereinis a first antibody that competes with an antibody described herein forbinding to GITR (e.g., human GITR), wherein the first antibody competesfor binding in an assay comprising the following steps: (a) incubatingGITR-transfected cells with the first antibody in unlabeled form in acontainer; and (b) adding an antibody described herein in labeled formin the container and incubating the cells in the container; and (c)detecting the binding of the antibody described herein in labeled formto the cells. In certain embodiments, provided herein is a firstantibody that competes with an antibody described herein for binding toGITR (e.g., human GITR), wherein the competition is exhibited as reducedbinding of the first antibody to GITR by more than 80% (e.g., 85%, 90%,95%, or 98%, or between 80% to 85%, 80% to 90%, 85% to 90%, or 85% to95%).

In specific aspects, provided herein is an antibody which competes(e.g., in a dose dependent manner) for specific binding to GITR (e.g.,human GITR), with an antibody comprising a VL domain having the aminoacid sequence selected from the group consisting of SEQ ID 202, 207,208, 400-411, 413-416, 418-421, 423-448, 450-452, 454-464, 467-477,481-486, 488-513, and 515-518, and a VH domain having the amino acidsequence selected from the group consisting of SEQ ID NO: 201, 206, 215,217-234, 236-256, 258, 259, 261-265, 267, 268, 271-273, 276, 277, 280,281, 283-285, 287, 288, 290, 291, 294, 296-299, 301, 304-306, 308,313-316, 319, 320, 322-325, 327, 328, 333, 336, 338-340, 342, 343, 350,354-356, 358-360, 362-368, 380, 384 and 387. In specific aspects,provided herein is an antibody which competes (e.g., in a dose dependentmanner) for specific binding to GITR (e.g., human GITR), with anantibody comprising a VL domain having the amino acid sequence selectedfrom the group consisting of SEQ ID 202, 207, 208, 400-411, 413-416,418-421, 423-448, 450-452, 454-464, 467-477, 481-486, 488-513, and515-519, and a VH domain having the amino acid sequence selected fromthe group consisting of SEQ ID NO: 201, 206, 215, 217-234, 236-256, 258,259, 261-265, 267, 268, 270-273, 276, 277, 280, 281, 283-285, 287, 288,290, 291, 294, 296-299, 301, 304-306, 308, 313-316, 319, 320, 322-325,327, 328, 333, 336, 338-340, 342, 343, 345, 350, 354-356, 358-360, and362-368.

In specific aspects, provided herein is an antibody which competes(e.g., in a dose dependent manner) for specific binding to GITR (e.g.,human GITR), with an antibody comprising (i) a VL domain comprising a VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequences of the VLCDRs of an antibody listed in Table 1; and (ii) a VH domain comprising aVH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of theCDRs of an antibody listed in Table 2 (e.g., the VH CDRs of a particularantibody referred to by name in Table 1, such as 231-32-15, Hum231#1, orHum231#2).

In a particular embodiment, provided herein is an antibody that competes(e.g., in a dose-dependent manner), for specific binding to GITR (e.g.,human GITR), with an antibody comprising the VH and VL CDRs of 231-32-15(SEQ ID NO: 201 and 202).

In a specific embodiment, an antibody described herein is one that iscompetitively blocked (e.g., in a dose dependent manner) by an antibodycomprising a VL domain having the amino acid sequence selected from thegroup consisting of SEQ ID NO: 202, 207, 208, 400-411, 413-416, 418-421,423-448, 450-452, 454-464, 467-477, 481-486, 488-513, and 515-518 and aVH domain having the amino acid sequence selected from the groupconsisting of SEQ ID NO: 201, 206, 215, 217-234, 236-256, 258, 259,261-265, 267, 268, 271-273, 276, 277, 280, 281, 283-285, 287, 288, 290,291, 294, 296-299, 301, 304-306, 308, 313-316, 319, 320, 322-325, 327,328, 333, 336, 338-340, 342, 343, 345, 350, 354-356, 358-360, 362-368,380, 384, and 387, for specific binding to GITR (e.g., human GITR). In aspecific embodiment, an antibody described herein is one that iscompetitively blocked (e.g., in a dose dependent manner) by an antibodycomprising a VL domain having the amino acid sequence selected from thegroup consisting of SEQ ID NO: 202, 207, 208, 400-411, 413-416, 418-421,423-448, 450-464, 467-477, 481-486, 488-513, and 515-519 and a VH domainhaving the amino acid sequence selected from the group consisting of SEQID NO: 201, 206, 215, 217-234, 236-256, 258, 259, 261-265, 267, 268,270-273, 276, 277, 280, 281, 283-285, 287, 288, 290, 291, 294, 296-299,301, 304-306, 308, 313-316, 319, 320, 322-325, 327, 328, 333, 336,338-340, 342, 343, 345, 350, 354-356, 358-360, and 362-368, for specificbinding to GITR (e.g., human GITR).

In one embodiment, an antibody described herein is one that iscompetitively blocked by an antibody comprising a VL domain having theamino acid sequence of SEQ ID NO: 207 or 208 and a VH domain having theamino acid sequence of SEQ ID NO: 206 for specific binding to GITR(e.g., human GITR).

In another specific embodiment, an antibody described herein is one thatis competitively blocked (e.g., in a dose dependent manner) by anantibody comprising (i) a VL domain comprising a VL CDR1, VL CDR2, andVL CDR3 having the amino acid sequences of the CDRs of antibody listedin Table 1 (e.g., the VL CDRs of a particular antibody referred by namein Table 1); and (ii) a VH domain comprising a VH CDR1, VH CDR2, and VHCDR3 having the amino acid sequences of the CDRs of antibody listed inTable 2.

In specific aspects, provided herein is an antibody, or anantigen-binding fragment thereof, which immunospecifically binds to thesame epitope as that of an antibody (e.g., any one of antibodiesHum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies 1-107, orantibodies pab2159, pab2160, pab2161, or Hum231#2w) comprising the aminoacid sequences described herein (see, e.g., Tables 1-4) for specificbinding to GITR (e.g., human GITR). Assays known to one of skill in theart or described herein (e.g., X-ray crystallography, ELISA assays,etc.) can be used to determine if two antibodies bind to the sameepitope.

In a specific embodiment, an antibody or an antigen-binding fragmentthereof described herein immunospecifically binds to the same epitope asthat of an antibody (e.g., any one of antibodies Hum231#1, Hum231#2,pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971,pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981,pab1983, 231-32-15 or antibodies 1-107) comprising a VL domain having anamino acid sequence selected from the group consisting of SEQ ID NO:202, 207, 208, 400-411, 413-416, 418-421, 423-448, 450-452, 454-464,467-477, 481-486, 488-513, and 515-518, and a VH domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 201, 206,215, 217-234, 236-256, 258, 259, 261-265, 267, 268, 271-273, 276, 277,280, 281, 283-285, 287, 288, 290, 291, 294, 296-299, 301, 304-306, 308,313-316, 319, 320, 322-325, 327, 328, 333, 336, 338-340, 342, 343, 345,350, 354-356, 358-360, 362-368, 380, 384, and 387). In a specificembodiment, an antibody or an antigen-binding fragment thereof describedherein immunospecifically binds to the same epitope as that of anantibody (e.g., any one of antibodies Hum231#1, Hum231#2, pab1964,pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972,pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15 or antibodies 1-107, or antibodies pab2159, pab2160, pab2161,or Hum231#2w) comprising a VL domain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 202, 207, 208, 400-411,413-416, 418-421, 423-448, 450-464, 467-477, 481-486, 488-513, and515-519, and a VH domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 201, 206, 215, 217-234, 236-256, 258,259, 261-265, 267, 268, 270-273, 276, 277, 280, 281, 283-285, 287, 288,290, 291, 294, 296-299, 301, 304-306, 308, 313-316, 319, 320, 322-325,327, 328, 333, 336, 338-340, 342, 343, 345, 350, 354-356, 358-360, and362-368).

In a specific embodiment, an antibody or an antigen-binding fragmentthereof described herein immunospecifically binds to the same epitope asthat bound by an antibody comprising the VH domain and VL domain ofantibody Hum231#1 or Hum231#2 (SEQ ID NOs: 206 and 207 or SEQ ID NOs:206 and 208, respectively), or an epitope that overlaps the epitope ofantibody comprising the VH domain and VL domain of antibody Hum231#1 orHum231#2 (SEQ ID NOs: 206 and 207 or SEQ ID NOs: 206 and 208,respectively).

In another specific embodiment, an antibody or an antigen-bindingfragment thereof described herein, immunospecifically binds to the sameepitope as that of an antibody comprising (i) a VL domain comprising aVL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of theCDRs of antibody listed in Table 1 (e.g., the VL CDRs of a particularantibody referred to by name in Table 1) and (ii) a VH domain comprisinga VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of theCDRs of antibody listed in Table 2 (e.g., the VH CDRs of a particularantibody referred to by name in Table 2).

In a particular embodiment, an antibody described herein or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR) and competitively blocks (e.g., in a dose dependentmanner) antibody 231-32-15, Hum231#1, Hum231#2 or Hum231#2w from bindingto GITR (e.g., human GITR), comprises a light chain variable region (VL)and a heavy chain variable region (VH), wherein

(i) the VL comprises:

(a) a VL CDR1 comprising the amino acid sequenceKSSQSX₁X₂X₃X₄X₅X₆X₇KX₈YLX₉ (SEQ ID NO: 4), wherein:

X₁ is L, A, V, I, P, F or M

X₂ is L, A, V, I, P, F, M or S

X₃ is N, G, Q, S, T, C, W, Y or A

X₄ is S, G, N, Q, T, C, W, Y or A

X₅ is G, N, Q, S, T, C, W, Y or A

X₆ is N, G, Q, S, T, C, W, Y or A

X₇ is Q, G, N, S, T, C, W, Y or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is T, G, N, Q, S, C, W, Y, V, I or A; and/or

(b) a VL CDR2 comprising the amino acid sequence X₁ASTRX₂X₃(SEQ ID NO:5), wherein:

X₁ is W, G, N, Q, S, T, C, Y, F, H or A

X₂ is E, D or A

X₃ is S, G, N, Q, T, C, W, Y or A; and/or

(c) a VL CDR3 comprising the amino acid sequence QX₁X₂YX₃X₄PYT (SEQ IDNO: 6), wherein:

X₁ is N, G, Q, S, T, C, W or Y

X₂ is D, E or Y

X₃ is S, G, N, Q, T, C, W, Y or A

X₄ is Y, G, N, Q, S, T, C, W, F, H, L, or A; and (ii) the VH comprises:

(a) a VH CDR1 comprising the amino acid sequence X₁YX₂MX₃ (SEQ ID NO:1), wherein

X₁ is D, E, G or A

X₂ is A, V, L, I, P, F, M or Y

X₃ is Y, G, N, Q, S, T, C, W, F or H; and/or

(b) a VH CDR2 comprising the amino acid sequenceX₁IX₂X₃X₄SGX₅X₆X₇YX₈QKFX₉X₁₀ (SEQ ID NO: 2), wherein

X₁ is V, A, L, I, P, F, M or T

X₂ is R, K, H, Q or A

X₃ is T, G, N, Q, S, C, W, Y, V, I or P

X₄ is Y, G, N, Q, S, T, C, W, F, H, or A

X₅ is D, E, G or A

X₆ is V, A, L, I, P, F, M or T

X₇ is T, G, N, Q, S, C, W, Y, V, I, P or A

X₈ is N, G, Q, S, T, C, W, Y or A

X₉ is K, R, H, Q or A

X₁₀ is D, E, G or A; and/or

(c) a VH CDR3 comprising the amino acid sequence SGTVRGX₁X₂X₃ (SEQ IDNO: 3), wherein

X₁ is F, A, V, L, I, P, M, Y, W, H or S

X₂ is A, or D

X₃ is Y, G, N, Q, S, T, C, W, F, H or V.

In a particular embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to GITR(e.g., human GITR) and competitively blocks (e.g., in a dose dependentmanner) antibody 231-32-15, Hum231#1, Hum231#2 or Hum231#2w from bindingto GITR (e.g., human GITR), comprises a light chain variable region (VL)and a heavy chain variable region (VH), wherein

(i) the VL comprises:

(a) a VL CDR1 comprising the amino acid sequence KSSQSLLNSX₁NQKNYLX₂(SEQ ID NO: 10), wherein

X₁ is G or S

X₂ is T or S; and/or

(b) a VL CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO:11); and/or

(c) a VL CDR3 comprising the amino acid sequence QNX₁YSX₂PYT (SEQ ID NO:12), wherein

X₁ is D or E

X₂ is Y, F or S and (ii) the VH comprises:

(a) a VH CDR1 comprising the amino acid sequence X₁YX₂MX₃ (SEQ ID NO:7), wherein

X₁ is D, E or G

X₂ is A or V

X₃ is Y or H; and/or

(b) a VH CDR2 comprising the amino acid sequenceX₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈ (SEQ ID NO: 8), wherein

X₁ is V or L

X₂ is R, K or Q

X₃ is Y or F

X₄ is D, E or G

X₅ is V or L

X₆ is T or S

X₇ is K, R or Q

X₈ is D, E or G; and/or

(c) a VH CDR3 comprising the amino acid sequence SGTVRGFAY (SEQ ID NO:9).

In some embodiments, an antibody that competes for binding with anantibody described herein for binding GITR (e.g., human GITR) or bindsto the same or an overlapping epitope of an antibody described herein(e.g., Hum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15, or antibodies 1-107, orantibodies pab2159, pab2160, pab2161, or Hum231#2w) prevents binding ofGITRL (e.g., human GITRL) to GITR (e.g., human GITR) by less than 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10%as assessed by an assay known to one of skill in the art or describedherein. In a specific embodiment, an antibody or antigen-bindingfragment thereof, which competes with an antibody described herein forbinding to GITR (e.g., human GITR) or binds to the same epitope oroverlapping epitope of an antibody described herein, inhibits binding ofGITRL (e.g., human GITRL) to GITR (e.g., human GITR) by less than 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10%as assessed by the assay described in Example 2, infra (e.g., Section6.2.5.2 or 6.2.5.4, infra). In another specific embodiment, an antibodyor antigen-binding fragment thereof, which competes with an antibodydescribed herein for binding to GITR (e.g., human GITR) or binds to thesame epitope or overlapping epitope of an antibody described herein, ata concentration of 1000 ng/ml, 950 ng/ml, 900 ng/ml, 850 ng/ml, 800ng/ml, 750 ng/ml, 700 ng/ml, 650 ng/ml, 600 ng/ml, 550 ng/ml, 500 ng/ml,450 ng/ml, 400 ng/ml, 350 ng/ml, 333 ng/ml, 300 ng/ml, 250 ng/ml, 200ng/ml, 100 ng/ml, 50 ng/ml or 10 ng/ml inhibits binding of 1.5 nM, 1.4nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL (e.g., GITRL-PE)to GITR coupled to beads (e.g., human GITR coupled to Luminex® beads) ata concentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/mlor 3 pg/ml per bead by less than 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%,45%, 40%, 35%, 30%, 25%, 20% or 10% relative to the binding of 1.5 nM,1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM,0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL to the GITRcoupled beads at a concentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml,5 pg/ml, 4 pg/ml or 3 pg/ml per bead in the absence of the anti-GITRantibody or antigen-binding fragment thereof in a suspension array assay(e.g., Luminex® 200 system). In another specific embodiment, an antibodyor antigen-binding fragment thereof, which competes with an antibodydescribed herein for binding to GITR (e.g., human GITR) or binds to thesame epitope or overlapping epitope of an antibody described herein, atconcentration of 1000 ng/ml to 750 ng/ml, 1000 ng/ml to500 ng/ml, 850ng/ml to 500 ng/ml, 750 ng/ml to 500 ng/ml, 600 ng/ml to 500 ng/ml, 500ng/ml to 400 ng/ml, 400 ng/ml to 300 ng/ml, or 300 ng/ml to 200 ng/mlinhibits binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM oflabeled GITRL (e.g., GITRL-PE) to GITR coupled to beads (e.g., humanGITR coupled to Luminex® beads) at a concentration of 9 pg/ml, 8 pg/ml,7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead by less than 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10%relative to the binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM,0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nMof labeled GITRL to the GITR coupled beads at a concentration of 9pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay (e.g., Luminex® 200 system).

In another specific embodiment, an antibody or antigen-binding fragmentthereof, which competes with an antibody described herein for binding toGITR (e.g., human GITR) or binds to the same epitope or overlappingepitope of an antibody described herein, at concentration of 3500 ng/ml,3400 ng/ml, 3300 ng/ml, 3200 ng/ml, 3100 ng/ml, 3000 ng/ml, 2900 ng/ml,2800 ng/ml, 2700 ng/ml, 2600 ng/ml, 2500 ng/ml, 2400 ng/ml, 2300 ng/ml,2200 ng/ml, 2100 ng/ml, 2000 ng/ml, 1900 ng/ml, 1800 ng/ml, 1700 ng/ml,1600 ng/ml, 1500 ng/ml, 1400 ng/ml, 1300 ng/ml, 1200 ng/ml, or 1100ng/ml inhibits binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM,0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nMof labeled GITRL (e.g., GITRL-PE) to GITR coupled to beads (e.g., humanGITR coupled to Luminex® beads) at a concentration of 9 pg/ml, 8 pg/ml,7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead by less than 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10%relative to the binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM,0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nMof labeled GITRL to the GITR coupled beads at a concentration of 9pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay (e.g., Luminex® 200 system). Inanother specific embodiment, an antibody or antigen-binding fragmentthereof, which competes with an antibody described herein for binding toGITR (e.g., human GITR) or binds to the same epitope or overlappingepitope of an antibody described herein, at concentration of 3500 ng/mlto 3200 ng/ml, 3500 ng/ml to 3000 ng/ml, 3200 ng/ml to 2500 ng/ml, 3000to 2200 ng/ml, 2500 ng/ml to 1800 ng/ml, 2000 ng/ml to 1500 ng/ml, 1700ng/ml to 1200 ng/ml, or 1500 ng/ml to 1000 ng/ml inhibits binding of 1.5nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL (e.g.,GITRL-PE) to GITR coupled to beads (e.g., human GITR coupled to Luminex®beads) at a concentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5pg/ml, 4 pg/ml or 3 pg/ml per bead by less than 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% relative to thebinding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM,0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeledGITRL to the GITR coupled beads at a concentration of 9 pg/ml, 8 pg/ml,7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead in the absence ofthe anti-GITR antibody or antigen-binding fragment thereof in asuspension array assay (e.g., Luminex® 200 system).

In certain embodiments, an antibody, which competes for binding with anantibody described herein for binding GITR (e.g., human GITR) or bindsto the same or an overlapping epitope of an antibody described herein,at a concentration of 3000 ng/ml prevents binding of 0.5 nM GITRL (e.g.,human GITRL) to GITR (e.g., human GITR) by less than 85% or less than80% when GITR (e.g., human GITR) is coupled to beads (e.g., Luminex®beads) at a concentration of 5 pg/ml per bead relative to the binding of0.5 nM of labeled GITRL to GITR coupled beads at a concentration of 5pg/ml/bead in the absence of the anti-GITR antibody or antigen-bindingfragment thereof in a suspension array assay (e.g., Luminex® 200system). In certain embodiments, an antibody, which competes for bindingwith an antibody described herein for binding GITR (e.g., human GITR) orbinds to the same or an overlapping epitope of an antibody describedherein, at a concentration of 1000 ng/ml prevents binding of 0.5 nMGITRL (e.g., human GITRL) to GITR (e.g., human GITR) by less than 85%,less than 80% or less than 75% when GITR (e.g., human GITR) is coupledto beads (e.g., Luminex® beads) at a concentration of 5 pg/ml per beadrelative to the binding of 0.5 nM of labeled GITRL to GITR coupled beadsat a concentration of 5 pg/ml/bead in the absence of the anti-GITRantibody or antigen-binding fragment thereof in a suspension array assay(e.g., Luminex® 200 system). In certain embodiments, an antibody, whichcompetes for binding with an antibody described herein for binding GITR(e.g., human GITR) or binds to the same or an overlapping epitope of anantibody described herein, at a concentration of 333 ng/ml preventsbinding of 0.5 nM GITRL (e.g., human GITRL) to GITR (e.g., human GITR)by less than 70% or less than 65% when GITR (e.g., human GITR) iscoupled to beads (e.g., Luminex® beads) at a concentration of 5 pg/mlper bead relative to the binding of 0.5 nM of labeled GITRL to GITRcoupled beads at a concentration of 5 pg/ml/bead in the absence of theanti-GITR antibody or antigen-binding fragment thereof in a suspensionarray assay (e.g., Luminex® 200 system). In certain embodiments, anantibody, which competes for binding with an antibody described hereinfor binding GITR (e.g., human GITR) or binds to the same or anoverlapping epitope of an antibody described herein, at a concentrationof 111 ng/ml prevents binding of 0.5 nM GITRL (e.g., human GITRL) toGITR (e.g., human GITR) by less than 65%, less than 60% or less than 55%when GITR (e.g., human GITR) is coupled to beads (e.g., Luminex® beads)at a concentration of 5 pg/ml per bead relative to the binding of 0.5 nMof labeled GITRL to GITR coupled beads at a concentration of 5pg/ml/bead in the absence of the anti-GITR antibody or antigen-bindingfragment thereof in a suspension array assay (e.g., Luminex® 200system). In certain embodiments, an antibody, which competes for bindingwith an antibody described herein for binding GITR (e.g., human GITR) orbinds to the same or an overlapping epitope of an antibody describedherein, at a concentration of 37 ng/ml prevents binding of 0.5 nM GITRL(e.g., human GITRL) to GITR (e.g., human GITR) by less than 40% whenGITR (e.g., human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml per bead relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay (e.g., Luminex® 200 system). Incertain embodiments, an antibody, which competes for binding with anantibody described herein for binding GITR (e.g., human GITR) or bindsto the same or an overlapping epitope of an antibody described herein,at a concentration of 12 ng/ml prevents binding of 0.5 nM GITRL (e.g.,human GITRL) to GITR (e.g., human GITR) by less than 20% when GITR(e.g., human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml per bead relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay (e.g., Luminex® 200 system).

In another embodiment, a certain amount of labeled GITRL (e.g., humanGITRL-PE) binds to GITR coupled to beads (e.g., human GITR coupled toLuminex® beads) in the presence of an antibody, which competes forbinding with an antibody described herein for binding to GITR or bindsto the same or an overlapping epitope of an antibody described herein,in a method comprising: (a) coupling GITR (e.g., human GITR) to beads(e.g., human GITR coupled to Luminex® beads) at a concentration of 5pg/ml per bead; (b) incubating the GITR coupled beads at a concentrationof 40 beads/μl with 3000 ng/ml, 2500 ng/ml, 2000 ng/ml, 1500 ng/ml, 1000ng/ml, 750 ng/ml, 500 ng/ml, 250 ng/ml, 100 ng/ml, 50 ng/ml or 10 ng/mlof the competing antibody or the antibody that binds to the same oroverlapping epitope in a well for a first period of time (e.g., 30minutes, 60 minutes, 1.5 hours, 2 hours, 2.5 hours or 3 hours), whereinthe well contains 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300,1400 or 1500 beads; (c) adding labeled GITRL (e.g., human GITRL-PE) tothe well to obtain a final concentration of 1.5 nM, 1.4 nM, 1.3 nM, 1.2nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3nM, 0.2 nM or 0.1 nM (in specific embodiments, 0.5 nM) of the labeledGITRL and 20 beads/μl of the GITR coupled beads, and incubating for asecond period of time (e.g., 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5hours or 3 hours); and (d) detecting the labeled GITRL bound to the GITRcoupled beads in, e.g., a suspension array assay such as the Luminex®200 system. In specific embodiments, the amount of the labeled GITRLbound to the GITR coupled beads in the presence of the competingantibody or the antibody that binds to the same or overlapping epitopeis determined relative to the amount of labeled GITRL bound to the GITRcoupled beads in the absence of the competing antibody or the antibodythat binds to the same or overlapping epitope. In certain embodiments,the absence of the competing antibody or the antibody that binds to thesame or overlapping epitope means that no antibody or antigen-bindingfragment thereof is present in the well. In other embodiments, theabsence of the competing antibody or the antibody that binds to the sameor overlapping epitope means that an isotype control antibody that doesnot bind to GITR is present in the well. In accordance with theseembodiments, the amount of labeled GITRL bound to the GITR coupled beadsin the presence of the competing antibody or the antibody that binds tothe same or overlapping epitope is determined to be, in someembodiments, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60%, or20% to 60%, 30% to 50%, or 20% to 70% of the amount of the labeled GITRLbound to the GITR coupled beads in the absence of the competing antibodyor the antibody that binds to the same or overlapping epitope.

In certain embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, or 75% of GITRL (e.g., human GITRL) binds toGITR (e.g., human GITR) in the presence of an antibody orantigen-binding fragment thereof, which competes with an antibodydescribed herein for binding to GITR (e.g., human GITR) or binds to thesame epitope or overlapping epitope of an antibody described herein,assessed by an assay known to one of skill in the art or describedherein. In a specific embodiment, at least 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of GITRL (e.g., human GITRL)binds to GITR (e.g., human GITR) in the presence of an antibody orantigen-binding fragment thereof, which competes with an antibodydescribed herein for binding to GITR (e.g., human GITR) or binds to thesame epitope or overlapping epitope of an antibody described herein, asassessed by the assay described in Example 2, infra (e.g., Sections6.2.5.2 or 6.2.5.4, infra). In another specific embodiment, at least10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM,0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeled GITRL (e.g.,labeled human GITRL, such as hGITRL-PE) binds to GITR coupled to beads(e.g., human GITR coupled to Luminex® beads) at a concentration of 9pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per beadin the presence of 1000 ng/ml, 950 ng/ml, 900 ng/ml, 850 ng/ml, 800ng/ml, 750 ng/ml, 700 ng/ml, 650 ng/ml, 600 ng/ml, 550 ng/ml, 500 ng/ml,450 ng/ml, 400 ng/ml, 350 ng/ml, 333 ng/ml, 300 ng/ml, 250 ng/ml, 200ng/ml, 100 ng/ml, 50 ng/ml or 10 ng/ml of an antibody or antigen-bindingfragment thereof, which competes with an antibody described herein forbinding to GITR (e.g., human GITR) or binds to the same epitope oroverlapping epitope of an antibody described herein, relative to thebinding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, 0.8 nM,0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM of labeledGITRL to the GITR coupled beads at a concentration of 9 pg/ml, 8 pg/ml,7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per bead in the absence ofthe anti-GITR antibody or antigen-binding fragment thereof in asuspension array assay (e.g., Luminex® 200 system). In another specificembodiment, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, or 75% of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM,0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nMof labeled GITRL (e.g., labeled human GITRL, such as hGITRL-PE) binds toGITR coupled to beads (e.g., human GITR coupled to Luminex® beads) at aconcentration of 9 pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or3 pg/ml per bead in the presence of 3500 ng/ml, 3400 ng/ml, 3300 ng/ml,3200 ng/ml, 3100 ng/ml, 3000 ng/ml, 2900 ng/ml, 2800 ng/ml, 2700 ng/ml,2600 ng/ml, 2500 ng/ml, 2400 ng/ml, 2300 ng/ml, 2200 ng/ml, 2100 ng/ml,2000 ng/ml, 1900 ng/ml, 1800 ng/ml, 1700 ng/ml, 1600 ng/ml, 1500 ng/ml,1400 ng/ml, 1300 ng/ml, 1200 ng/ml, or 1100 ng/ml of an antibody orantigen-binding fragment thereof, which competes with an antibodydescribed herein for binding to GITR (e.g., human GITR) or binds to thesame epitope or overlapping epitope of an antibody described herein,relative to the binding of 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM,0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nMof labeled GITRL to the GITR coupled beads at a concentration of 9pg/ml, 8 pg/ml, 7 pg/ml, 6 pg/ml, 5 pg/ml, 4 pg/ml or 3 pg/ml per beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay (e.g., Luminex® 200 system).

In certain embodiments, an antibody, which competes for binding with anantibody described herein for binding GITR (e.g., human GITR) or bindsto the same or an overlapping epitope of an antibody described herein,at a concentration of 3000 ng/ml does not prevent binding of 0.5 nMGITRL (e.g., human GITRL) to GITR (e.g., human GITR) by more than 15% ormore than 20% when GITR (e.g., human GITR) is coupled to beads (e.g.,Luminex® beads) at a concentration of 5 pg/ml per bead relative to thebinding of 0.5 nM of labeled GITRL to GITR coupled beads at aconcentration of 5 pg/ml/bead in the absence of the anti-GITR antibodyor antigen-binding fragment thereof in a suspension array assay (e.g.,Luminex® 200 system). In certain embodiments, an antibody, whichcompetes for binding with an antibody described herein for binding GITR(e.g., human GITR) or binds to the same or an overlapping epitope of anantibody described herein, at a concentration of 1000 ng/ml does notprevent binding of 0.5 nM GITRL (e.g., human GITRL) to GITR (e.g., humanGITR) by more than 15%, more than 20% or more than 25% when GITR (e.g.,human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml per bead relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay (e.g., Luminex® 200 system). Incertain embodiments, an antibody, which competes for binding with anantibody described herein for binding GITR (e.g., human GITR) or bindsto the same or an overlapping epitope of an antibody described herein,at a concentration of 333 ng/ml does not prevent binding of 0.5 nM GITRL(e.g., human GITRL) to GITR (e.g., human GITR) by more than 30% or morethan 35% when GITR (e.g., human GITR) is coupled to beads (e.g.,Luminex® beads) at a concentration of 5 pg/ml per bead relative to thebinding of 0.5 nM of labeled GITRL to GITR coupled beads at aconcentration of 5 pg/ml/bead in the absence of the anti-GITR antibodyor antigen-binding fragment thereof in a suspension array assay (e.g.,Luminex® 200 system). In certain embodiments, an antibody, whichcompetes for binding with an antibody described herein for binding GITR(e.g., human GITR) or binds to the same or an overlapping epitope of anantibody described herein, at a concentration of 111 ng/ml does notprevent binding of 0.5 nM GITRL (e.g., human GITRL) to GITR (e.g., humanGITR) by more than 35%, more than 40% or more than 45% when GITR (e.g.,human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml per bead relative to the binding of 0.5 nM oflabeled GITRL to GITR coupled beads at a concentration of 5 pg/ml/beadin the absence of the anti-GITR antibody or antigen-binding fragmentthereof in a suspension array assay (e.g., Luminex® 200 system). Incertain embodiments, an antibody, which competes for binding with anantibody described herein for binding GITR (e.g., human GITR) or bindsto the same or an overlapping epitope of an antibody described herein,at a concentration of 37 ng/ml does not prevent binding of 0.5 nM GITRL(e.g., human GITRL) to GITR (e.g., human GITR) by more than 60% whenGITR (e.g., human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml relative to the binding of 0.5 nM of labeledGITRL to GITR coupled beads at a concentration of 5 pg/ml/bead in theabsence of the anti-GITR antibody or antigen-binding fragment thereofper bead in a suspension array assay (e.g., Luminex® 200 system). Incertain embodiments, an antibody, which competes for binding with anantibody described herein for binding GITR (e.g., human GITR) or bindsto the same or an overlapping epitope of an antibody described herein,at a concentration of 12 ng/ml does not prevent binding of 0.5 nM GITRL(e.g., human GITRL) to GITR (e.g., human GITR) by more than 85% whenGITR (e.g., human GITR) is coupled to beads (e.g., Luminex® beads) at aconcentration of 5 pg/ml relative to the binding of 0.5 nM of labeledGITRL to GITR coupled beads at a concentration of 5 pg/ml/bead in theabsence of the anti-GITR antibody or antigen-binding fragment thereofper bead in a suspension array assay (e.g., Luminex® 200 system).

In certain embodiments, provided herein is an antibody, which competesfor binding with an antibody described herein for binding GITR (e.g.,human GITR) or binds to the same or an overlapping epitope of anantibody described herein, at a concentration of 150 nM, 145 nM, 140 nM,135 nM, 130 nM, 125 nM, 120 nM, 115 nM, 110 nM, 105 nM or 100 nM boundto GITR (e.g., human GITR) immobilized on a chip (e.g., CM5 sensor chip)inhibits binding of 150 nM, 145 nM, 140 nM, 135 nM, 130 nM, 125 nM, 120nM, 115 nM, 110 nM, 105 nM or 100 nM of GITRL (e.g., non-covalentlylinked trimer of human GITRL) to the GITR immobilized on the chip byless than 60%, less than 55%, less than 50%, less than 45%, less than40%, less than 35%, less than 30%, less than 25%, less than 20% or lessthan 15%. In certain embodiments, provided herein is an antibody, whichcompetes for binding with an antibody described herein for binding GITR(e.g., human GITR) or binds to the same or an overlapping epitope of anantibody described herein, wherein the competing antibody or theantibody that binds to the same or overlapping epitope at aconcentration of 125 nM bound to GITR (e.g., human GITR) immobilized ona chip (e.g., CM5 sensor chip) inhibits binding of 125 nM of GITRL(e.g., non-covalently linked trimer of human GITRL) to the GITRimmobilized on the chip by less than 60%, less than 55%, less than 50%,less than 45%, less than 40%, less than 35%, less than 30%, less than25%, less than 20% or less than 15%.

In certain embodiments, an antibody or fragment thereof, which competeswith an antibody described herein for binding to GITR (e.g., human GITR)or binds to the same epitope or overlapping epitope of an antibodydescribed herein, binds to GITR (e.g., human GITR) with a dissociationrate constant (k_(off)) of 8.5×10⁻³ s⁻¹ or less, 3.5×10⁻³ s⁻¹ or less,5×10⁻³ s⁻¹ or less, 2.5×10⁻³ s⁻¹ or less, 1×10⁻³ s⁻¹ or less, 8.5×10⁻⁴s⁻¹ or less, 5×10⁻⁴ s⁻¹ or less, 3.5×10⁻⁴ s⁻¹ or less, 2.5×10⁻⁴ s⁻¹ orless, 1×10⁻⁴ s⁻¹ or less, 8.5×10⁻⁵⁻ s⁻¹ or less, 3.5×10⁻⁵⁻ s⁻¹ or less,5×10⁻⁵⁻ s⁻¹ or less, 2.5×10⁻⁵⁻ s⁻¹ or less, 1×10⁻⁵⁻ s⁻¹ or less,8.5×10⁻⁶⁻ s⁻¹ or less, 5×10⁻⁶⁻ s⁻¹ or less, 3.5×10⁻⁶⁻ s⁻¹ or less,2.5×10⁻⁶⁻ s⁻¹ or less, 1×10⁻⁶⁻ s⁻¹ or less, 8.5×10⁻⁷⁻ s⁻¹ or less,5×10⁻⁷⁻ s⁻¹ or less, 2.5×10⁻⁷⁻ s⁻¹ or less, 1×10⁻⁷⁻ s⁻¹ or less,8.5×10⁻⁸⁻ s⁻¹ or less, 5×10⁻⁸⁻ s⁻¹ or less, 2.5×10⁻⁸⁻ s⁻¹ or less,1×10⁻⁸⁻ s⁻¹ or less, 8.5×10⁻⁹⁻ s⁻¹ or less, 5×10⁻⁹ s⁻¹ or less,2.5×10⁻⁹⁻ s⁻¹ or less, or 1×10⁻⁹⁻ s⁻¹ or less. In some embodiments, anantibody or fragment thereof, which competes with an antibody describedherein for binding to GITR (e.g., human GITR) or binds to the sameepitope or overlapping epitope of an antibody described herein, binds toGITR (e.g., human GITR) with a k_(off) of between 9.5×10⁻⁵⁻ s⁻¹ to1×10⁻⁹⁻ s⁻¹, 8.5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁹⁻ s⁻¹, 5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁹⁻ s⁻¹,9.5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁸⁻ s⁻¹, 5×10⁻⁵ s⁻¹ to 1×10⁻⁸⁻ s⁻¹, 9.5×10⁻⁵⁻ s⁻¹to 1×10⁻⁷⁻ s⁻¹, 5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁷⁻ s⁻¹, 9.5×10⁻⁵⁻ s⁻¹ to 5×10⁻⁶⁻s⁻¹, 9.5×10⁻⁵⁻ s⁻¹ to 1×10⁻⁵⁻ s⁻¹, 8.5×10⁻³ s⁻¹ to 1×10⁻⁴ s⁻¹, 5×10⁻³s⁻¹ to 2.5×10⁻⁴ s⁻¹, 8.5×10⁻³ s⁻¹, to 1×10⁻⁵ s⁻¹, 8.5×10⁻⁵⁻ s⁻¹ to5×10⁻⁵⁻ s⁻¹. In certain embodiments, the k_(off) is determined using amonovalent antibody, such as a Fab fragment, as measured by, e.g.,BIAcore® surface plasmon resonance technology. In other embodiments, thek_(off) is determined using a bivalent antibody as measured by, e.g.,BIAcore® surface plasmon resonance technology. In a particularembodiment, the k_(off) is determined using an assay described inSection 6, infra.

In certain embodiments, an antibody or fragment thereof, which competeswith an antibody described herein for binding to GITR (e.g., human GITR)or binds to the same epitope or overlapping epitope of an antibodydescribed herein, binds to GITR (e.g., human GITR) with an associationrate constant (k_(on)) of at least 10⁵ M⁻¹s⁻¹, at least 2.5×10⁵ M⁻¹s⁻¹,at least 3.5×10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻s⁻¹, at least 10⁶ M⁻¹s⁻¹, atleast 2.5×10⁶ M⁻¹s⁻¹, at least 3.5×10⁶ M⁻¹s⁻¹, at least 5×10⁶ M⁻¹s⁻¹, atleast 10⁷ M⁻¹s⁻¹, at least 5×10⁷ M⁻¹s⁻¹, at least 10⁸ M⁻¹s⁻¹, at least 510⁸ M⁻¹s⁻¹ or at least 10⁹ M⁻¹s⁻¹. In some embodiments, an antibody orfragment thereof, which competes with an antibody described herein forbinding to GITR (e.g., human GITR) or binds to the same epitope oroverlapping epitope of an antibody described herein, binds to GITR(e.g., human GITR) with a k_(on) of between 1×10⁵ M⁻¹s⁻¹ to 5×10⁵M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹ to 1×10⁶ M⁻¹s⁻¹, 3.5×10⁵ M⁻¹s⁻¹ to 2.5×10⁶ M⁻¹s⁻¹,3.5×10⁵ M⁻¹s⁻¹ to 3.5×10⁶ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹ to 5×10⁶ M⁻¹s⁻¹, 1×10⁵M⁻¹s⁻¹ to 1×10⁷ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹ to 5×10⁷ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹ to10⁸ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹ to 1×10⁹ M⁻¹s⁻¹, 1×10⁶ M⁻¹s⁻¹ to 1×10⁷ M⁻¹s⁻¹,1×10⁶ M⁻¹s⁻¹ to 1×10⁸ M⁻¹s⁻¹, 1×10⁶ M⁻¹s⁻¹ to 1×10⁹ M⁻¹s⁻¹, 1×10⁷ M⁻¹s⁻¹to 1×10⁸ M⁻¹s⁻¹, 1×10⁷ M⁻¹s⁻¹ to 1×10⁹ M⁻¹s⁻¹, 1×10⁸ M⁻¹s⁻¹ to 1×10⁹M⁻¹s⁻¹. In certain embodiments, the k_(on) is determined using amonovalent antibody, such as a Fab fragment, as measured by, e.g.,BIAcore® surface plasmon resonance technology. In other embodiments, thek_(on) is determined using a bivalent antibody as measured by, e.g.,BIAcore® surface plasmon resonance technology. In a particularembodiment, the k_(on) is determined using an assay described in Section6, infra.

In certain embodiments, an antibody or fragment thereof, which competeswith an antibody described herein for binding to GITR (e.g., human GITR)or binds to the same epitope or overlapping epitope of an antibodydescribed herein, binds to GITR (e.g., human GITR) with a K_(D) of lessthan 7 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM,1 nM, 0.75 nM, 0.5 nM, 0.25 nM, or 0.1 nM. In some embodiments, anantibody or fragment thereof, which competes with an antibody describedherein for binding to GITR (e.g., human GITR) or binds to the sameepitope or overlapping epitope of an antibody described herein, binds toGITR (e.g., human GITR) with a K_(D) of about 7 nM, 6 nM, 5 nM, 4.5 nM,4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.75 nM, 0.5 nM, 0.25nM, or 0.1 nM. In certain embodiments, an antibody or fragment thereofdescribed herein, which competes with an antibody described herein forbinding to GITR (e.g., human GITR) or binds to the same epitope oroverlapping epitope of an antibody described herein, binds to GITR(e.g., human GITR) with a K_(D) of 7 nM to 4 nM, 7 nM to 5 nM, 6 nM to 4nM, 5 nM to 3 nM, 5 nM to 1 nM, 5 nM to 0.5 nM, 4 nM to 3 nM, 4 nM to 2nM, 4 nM to 1 nM, 4 nM to 0.5 nM, 3 nM to 2 nM, 3 nM to 1 nM, 3 nM to0.5 nM, 2 nM to 1 nM, 2 nM to 0.5 nM, 3 nM to 0.1 nM, 2 nM to 0.1 nM, 1nM to 0.1 nM, or 0.5 nM to 0.1 nM. In certain embodiments, the K_(D) iscalculated as the quotient of k_(off)/k_(on), and the k_(on) and k_(off)are determined using a monovalent antibody, such as a Fab fragment, asmeasured by, e.g., BIAcore® surface plasmon resonance technology. Inother embodiments, the K_(D) is calculated as the quotient ofk_(off)/k_(on), and the k_(on) and k_(off) are determined using abivalent antibody, such as a Fab fragment, as measured by, e.g.,BIAcore® surface plasmon resonance technology. In a specific embodiment,the K_(D) is determined as set forth in the Examples in Section 6, infra(e.g., Example 2).

In certain embodiments, the epitope of an antibody described herein isused as an immunogen to produce antibodies. See, e.g., Section 5.2 infrafor methods for producing antibodies.

In specific aspects, an antibody or fragment thereof described, whichspecifically binds to GITR (e.g., human GITR), does not inhibit (e.g.,in a dose dependent manner) the binding of the murine antibody 6C8 toGITR (e.g., human GITR) in an assay known to one of skill in the art ordescribed herein. See, e.g., U.S. Pat. No. 7,812,135 for a descriptionof the murine antibody 6C8. In certain embodiments, at least 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore of the murine antibody 6C8 binds to GITR (e.g., human GITR) in thepresence of an antibody or fragment thereof described, whichspecifically binds to GITR (e.g., human GITR), as assessed in an assayknown to one of skill in the art or described herein. In a specificembodiment, an antibody or fragment thereof described, whichspecifically binds to GITR (e.g., human GITR), does not inhibit (e.g.,in a dose dependent manner) the binding of the murine antibody 6C8 toGITR (e.g., human GITR) as assessed in the assay described in Example 6,infra. In certain embodiments, at least 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the murineantibody 6C8 binds to GITR (e.g., human GITR) in the presence of anantibody or fragment thereof described, which specifically binds to GITR(e.g., human GITR), as assessed in the assay described in Example 6,infra.

In some embodiments, anti-GITR antibodies described herein may bemultispecific antibodies, e.g., bispecific antibodies. In a particularembodiment, an anti-GITR antibody described herein is a bispecificantibody, wherein the antibody has specificities for at least twodifferent, typically non-overlapping epitopes. In a particularembodiment, a bispecific antibody comprises one arm comprising of anantibody described herein with specificity for GITR (e.g., human GITR),and a second arm comprising an antibody with specificity for a differentepitope on GITR (e.g., human GITR) or an epitope on a differentmolecule, e.g., PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3 or OX40. Forexample, the bispecific antibody may comprise one arm comprising anantibody described herein with specificity for GITR (e.g., human GITR)and a second arm comprising an antibody with specificity for CTLA-4,such as tremelimumab (Pfizer), ipilimumab (Yervoy®, Bristol-MeyersSquibb), an antibody that binds to the same epitope as tremelimumab oran overlapping epitope thereto, or an antibody that binds to the sameepitope as ipilimumab or an overlapping epitope thereto.

In specific aspects, an antibody or fragment thereof described herein,which immunospecifically binds to GITR (e.g., human GITR), functions asan agonist.

In certain embodiments, an antibody or fragment thereof describedherein, which immunospecifically binds to GITR (e.g., human GITR),increases GITR (e.g., human GITR) activity by at least about 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold as assessed by methods described herein and/or knownto one of skill in the art, relative to GITR (e.g., human GITR) activityin the presence or absence of GITRL (e.g., human GITRL) stimulationwithout any antibody or with an unrelated antibody (e.g., an antibodythat does not immunospecifically bind to GITR). In certain embodiments,an antibody or fragment thereof described herein, whichimmunospecifically binds to GITR (e.g., human GITR), increases GITR(e.g., human GITR) activity by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or99% as assessed by methods described herein and/or known to one of skillin the art, relative to GITR (e.g., human GITR) activity in the presenceor absence of GITRL (e.g., human GITRL) stimulation without any antibodyor with an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to GITR). Non-limiting examples of GITR (e.g.,human GITR) activity can include cell proliferation, GITR (e.g., humanGITR) signaling, cell survival, and cytokine production (e.g., IL-2,IL-6, IL-10, TNF-α, and IFN-γ). In specific embodiments, an antibody orfragment thereof described herein, which immunospecifically binds toGITR (e.g., human GITR), induces or increases a GITR (e.g., human GITR)activity together with GITRL (e.g., human GITRL). In certainembodiments, an antibody or fragment thereof described herein, whichimmunospecifically binds to GITR (e.g., human GITR), induces, enhances,or increases a GITR (e.g., human GITR) activity in the absence of GITRL(e.g., human GITRL). In specific embodiments, the antibody orantibody-binding fragment induces, enhances, or increases a GITRactivity and does not inhibit (e.g., does not completely inhibit or onlypartially inhibits) GITRL from binding to GITR. In specific embodiments,an increase in a GITR activity is assessed as described in the Examples,infra.

In certain aspects, an antibody or fragment thereof described herein,which immunospecifically binds to GITR (e.g., human GITR), induces,enhances, or increases the cellular proliferation of cells that expressGITR and that respond to GITR signaling (e.g., cells that proliferate inresponse to GITR stimulation and GITR signaling, such as T cells). Cellproliferation assays are described in the art, such as a ³H-thymidineincorporation assay, BrdU incorporation assay or CFSE assay, such asdescribed in Example 3, and can be readily carried out by one of skillin the art. In specific embodiments, T cells (e.g., CD4⁺ or CD8⁺effector T cells) stimulated with a T cell mitogen or T cell receptorcomplex stimulating agent (e.g., phytohaemagglutinin (PHA) and/orphorbol myristate acetate (PMA), or a TCR complex stimulating antibody,such as an anti-CD3 antibody and anti-CD28 antibody) in the presence ofan antibody or fragment thereof described herein, whichimmunospecifically binds to GITR (e.g., human GITR), have increasedcellular proliferation relative to T cells only stimulated with the Tcell mitogen or T cell receptor complex stimulating agentphytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody). See Example 3, infra, which demonstrates anincrease in T cell proliferation in the presence of an antibodydescribed herein that immunospecifically binds to GITR.

In one embodiment, CD8⁺ T cells stimulated with a T cell mitogen or Tcell receptor complex stimulating agent (e.g., phytohaemagglutinin (PHA)and/or phorbol myristate acetate (PMA), or a TCR complex stimulatingantibody, such as an anti-CD3 antibody and anti-CD28 antibody) in thepresence of an antibody or fragment thereof described herein, whichimmunospecifically binds to GITR (e.g., human GITR), have increasedcellular proliferation relative to T cells only stimulated with the Tcell mitogen or T cell receptor complex stimulating agent (e.g.,phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody). In another embodiment, CD4⁺ T cells stimulated witha T cell mitogen or T cell receptor complex stimulating agent (e.g.,phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody) in the presence of an antibody or fragment thereofdescribed herein, which immunospecifically binds to GITR (e.g., humanGITR), have increased cellular proliferation relative to T cells onlystimulated with the T cell mitogen or T cell receptor complexstimulating agent (e.g., phytohaemagglutinin (PHA) and/or phorbolmyristate acetate (PMA), or a TCR complex stimulating antibody, such asan anti-CD3 antibody and anti-CD28 antibody). In another embodiment,CD4⁺ T cells and CD8⁺ T cells stimulated with a T cell mitogen or T cellreceptor complex stimulating agent (e.g., phytohaemagglutinin (PHA)and/or phorbol myristate acetate (PMA), or a TCR complex stimulatingantibody, such as an anti-CD3 antibody and anti-CD28 antibody) in thepresence of an antibody or fragment thereof described herein, whichimmunospecifically binds to GITR (e.g., human GITR), have increasedcellular proliferation relative to T cells only stimulated with the Tcell mitogen or T cell receptor complex stimulating agent (e.g.,phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody). In some embodiments, T cells that have not beenstimulated with a T cell mitogen or T cell receptor complex stimulatingagent (e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate(PMA), or a TCR complex stimulating antibody, such as an anti-CD3antibody and anti-CD28 antibody) in the presence of an antibody orfragment thereof described herein, which immunospecifically binds toGITR (e.g., human GITR) have increased GITR activity and/or increasedNF-κB activity relative to T cells not in the presence of an antibody orfragment thereof described herein, which immunospecifically binds toGITR (e.g., human GITR).

In specific embodiments, an antibody or fragment thereof describedherein, which immunospecifically binds to GITR (e.g., human GITR),increases cell proliferation (e.g., T cells, such as CD4 and CD8effector T cells) by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, asassessed by methods described herein or known to one of skill in the art(e.g., ³H-thymidine incorporation assay, BrdU incorporation assay orCFSE assay, such as described in Example 3, infra), relative to GITR(e.g., human GITR) activity in the presence or absence of GITRL (e.g.,human GITRL) stimulation without any antibody or with an unrelatedantibody (e.g., an antibody that does not immunospecifically bind toGITR). In specific embodiments, an antibody or fragment thereofdescribed herein, which immunospecifically binds to GITR (e.g., humanGITR), increases cell proliferation (e.g., T cells, such as CD4 and CD8effector T cells) by at least at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99%, as assessed by methods described herein or known to one ofskill in the art (e.g., ³H-thymidine incorporation assay, BrdUincorporation assay or CFSE assay, such as described in Example 3,infra), relative to GITR (e.g., human GITR) activity in the presence orabsence of GITRL (e.g., human GITRL) stimulation without any antibody orwith an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to GITR).

In some embodiments, T cells (e.g., CD4⁺ or CD8⁺ effector T cells)stimulated with a T cell mitogen (e.g., an anti-CD3 antibody or phorbolester) in the presence of an antibody or fragment thereof describedherein, which immunospecifically binds to GITR (e.g., human GITR), haveincreased cellular proliferation by at least about 1.2 fold, 1.3 fold,1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold,30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100fold relative to T cells only stimulated with the T cell mitogen, asassessed by methods described herein or known to one of skill in the art(e.g., ³H-thymidine incorporation assay, BrdU incorporation assay orCFSE assay, such as described in Example 3, infra). In some embodiments,T cells (e.g., CD4⁺ or CD8⁺ effector T cells) stimulated with a T cellmitogen or T cell receptor complex stimulating agent (e.g.,phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody) in the presence of an antibody or fragment thereofdescribed herein, which immunospecifically binds to GITR (e.g., humanGITR), have increased cellular proliferation by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 99% relative to T cells only stimulated with theT cell mitogen or T cell receptor complex stimulating agent (e.g.,phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody), as assessed by methods described herein or known toone of skill in the art (e.g., ³H-thymidine incorporation assay, BrdUincorporation assay or CFSE assay, such as described in Example 3,infra). In a specific embodiment, cell proliferation is assessed asdescribed in Example 3, infra.

In certain aspects, an antibody or fragment thereof described herein,which immunospecifically binds to GITR (e.g., human GITR), increases thesurvival of cells (e.g., T cells, such as CD4 and CD8 effector T cells).In a specific embodiment, T cells (e.g., CD4⁺ or CD8⁺ effector T cells)stimulated with a T cell mitogen or T cell receptor complex stimulatingagent (e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate(PMA), or a TCR complex stimulating antibody, such as an anti-CD3antibody and anti-CD28 antibody) in the presence of an antibody orfragment thereof described herein, which immunospecifically binds toGITR (e.g., human GITR), have increased survival relative to T cellsonly stimulated with the T cell mitogen. Cell survival assays aredescribed in the art (e.g., a trypan blue exclusion assay) and can bereadily carried out by one of skill in the art.

In specific embodiments, an antibody or fragment thereof describedherein, which immunospecifically binds to GITR (e.g., human GITR),increases cell survival (e.g., T cells, such as CD4 and CD8 effector Tcells) by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold,2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed by methodsdescribed herein or known to one of skill in the art (e.g., a trypanblue exclusion assay), relative to cell survival in the presence orabsence of GITRL (e.g., human GITRL) stimulation without any antibody orwith an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to GITR). In specific embodiments, an antibodyor fragment thereof described herein, which immunospecifically binds toGITR (e.g., human GITR), increases cell survival (e.g., T cells, such asCD4 and CD8 effector T cells) by at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99%, as assessed by methods described herein or known to one ofskill in the art (e.g., a trypan blue exclusion assay), relative to cellsurvival in the presence or absence of GITRL (e.g., human GITRL)stimulation without any antibody or with an unrelated antibody (e.g., anantibody that does not immunospecifically bind to GITR).

In some embodiments, T cells (e.g., CD4⁺ or CD8⁺ effector T cells)stimulated with a T cell mitogen (e.g., an anti-CD3 antibody or phorbolester) in the presence of an antibody or fragment thereof describedherein, which immunospecifically binds to GITR (e.g., human GITR), haveincreased cell survival by at least about 1.2 fold, 1.3 fold, 1.4 fold,1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold,6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold relativeto T cells only stimulated with the T cell mitogen or T cell receptorcomplex stimulating agent (e.g., phytohaemagglutinin (PHA) and/orphorbol myristate acetate (PMA), or a TCR complex stimulating antibody,such as an anti-CD3 antibody and anti-CD28 antibody), as assessed bymethods described herein or known to one of skill in the art (e.g., atrypan blue exclusion assay). In some embodiments, T cells (e.g., CD4⁺or CD8⁺ effector T cells) stimulated with a T cell mitogen or T cellreceptor complex stimulating agent (e.g., phytohaemagglutinin (PHA)and/or phorbol myristate acetate (PMA), or a TCR complex stimulatingantibody, such as an anti-CD3 antibody and anti-CD28 antibody) in thepresence of an antibody or fragment thereof described herein, whichimmunospecifically binds to GITR (e.g., human GITR), have increased cellsurvival by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% relativeto T cells only stimulated with the T cell mitogen, as assessed bymethods described herein or known to one of skill in the art (e.g., atrypan blue exclusion assay).

In certain embodiments, an antibody or fragment thereof describedherein, which immunospecifically binds to GITR (e.g., human GITR),protects effector T cells (e.g., CD4⁺ and CD8⁺ effector T cells) fromactivation-induced cell death. In some embodiments, an antibody orfragment thereof described herein, which immunospecifically binds toGITR (e.g., human GITR), induces resistance of effector T cells (e.g.,CD4⁺ and CD8⁺ effector T cells) to Treg-mediated suppression.

In specific embodiments, an antibody or fragment thereof describedherein, which immunospecifically binds to GITR (e.g., human GITR),induces, enhances, or increases cytokine production (e.g., IL-2, IL-6,IL-10, TNF-α, and IFN-γ) by at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or99%, as assessed by methods described herein (see the Examples, infra,such as Example 3) or known to one of skill in the art, relative tocytokine production in the presence or absence of GITRL (e.g., humanGITRL) stimulation without any antibody or with an unrelated antibody(e.g., an antibody that does not immunospecifically bind to GITR). Inspecific embodiments, an antibody or fragment thereof described herein,which immunospecifically binds to GITR (e.g., human GITR), induces orenhances cytokine production (e.g., IL-2, IL-6, IL-10, TNF-α, and IFN-γ)by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed by methodsdescribed herein (see the Examples, infra, such as Example 3) or knownto one of skill in the art, relative to cytokine production in thepresence or absence of GITRL (e.g., human GITRL) stimulation without anyantibody or with an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to GITR).

In certain embodiments, T cells (e.g., CD4⁺ or CD8⁺ effector T cells)stimulated with a T cell mitogen or T cell receptor complex stimulatingagent (e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate(PMA), or a TCR complex stimulating antibody, such as an anti-CD3antibody and anti-CD28 antibody) in the presence of an antibody orfragment thereof described herein, which immunospecifically binds toGITR (e.g., human GITR), have increased cytokine production (e.g., IL-2,IL-6, IL-10, TNF-α, and IFN-γ) by at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99% relative to T cells only stimulated with the T cell mitogenor T cell receptor complex stimulating agent (e.g., phytohaemagglutinin(PHA) and/or phorbol myristate acetate (PMA), or a TCR complexstimulating antibody, such as an anti-CD3 antibody and anti-CD28antibody), as assessed by methods described herein or known to one ofskill in the art (e.g., an ELISA assay or as described in the Examples,infra). In some embodiments, T cells (e.g., CD4⁺ or CD8⁺ effector Tcells) stimulated with a T cell mitogen or T cell receptor complexstimulating agent (e.g., phytohaemagglutinin (PHA) and/or phorbolmyristate acetate (PMA), or a TCR complex stimulating antibody, such asan anti-CD3 antibody and anti-CD28 antibody) in the presence of anantibody or fragment thereof described herein, which immunospecificallybinds to GITR (e.g., human GITR), have increased cytokine production(e.g., IL-2, IL-6, IL-10, TNF-α, and IFN-γ) by at least about 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold relative to T cells only stimulated with the T cellmitogen or T cell receptor complex stimulating agent (e.g.,phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody), as assessed by methods described herein or known toone of skill in the art (e.g., an ELISA assay or as described in theExamples, infra).

In certain embodiments, an anti-GITR antibody or antigen bindingfragment thereof induces, enhances or activates an activity of GITR, inthe absence of a TCR agonist (e.g., an anti-CD3 antibody). GITR activitycan be assessed by measuring activation of canonical and non-canonicalNF-κB pathways. GITR activity can be assessed by measuring activation ofTRAF adapter mediated signaling pathways. The TRAF adapter is selectedfrom the group consisting of TRAF1, TRAF2, TRAF3, TRAF4, and TRAF5. GITRactivity can be assessed by measuring activation of MAPK/ERK pathway(also called the Ras-Raf-MEK-ERK pathway). Examples of “a TCR agonist”include, but are not limited to, antibodies targeting the T cellreceptor complex (e.g., an anti-CD3 antibody) and peptides bound tohuman leukocyte antigens, e.g., MHC class I and MHC class II, whereinthe peptides are derived from self, mutated self, or pathogen associatedproteins (e.g., viral or bacterial).

An anti-GITR antibody or antigen-binding fragment thereof can be fusedor conjugated (e.g., covalently or noncovalently linked) to a detectablelabel or substance. Examples of detectable labels or substances includeenzyme labels, such as, glucose oxidase; radioisotopes, such as iodine(¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In),and technetium (⁹⁹Tc); luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin. Suchlabeled antibodies or antigen-binding fragments can be used to detectGITR (e.g., human GITR) protein. See, e.g., Section 5.4.2, infra.

5.2 Antibody Production

Antibodies or fragments thereof that immunospecifically bind to GITR(e.g., human GITR) can be produced by any method known in the art forthe synthesis of antibodies, for example, by chemical synthesis or byrecombinant expression techniques. The methods described herein employs,unless otherwise indicated, conventional techniques in molecularbiology, microbiology, genetic analysis, recombinant DNA, organicchemistry, biochemistry, PCR, oligonucleotide synthesis andmodification, nucleic acid hybridization, and related fields within theskill of the art. These techniques are described, for example, in thereferences cited herein and are fully explained in the literature. See,e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989),Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel F M et al., Current Protocols in MolecularBiology, John Wiley & Sons (1987 and annual updates); Current Protocolsin Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.)(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press;Eckstein (ed.) (1991) Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: ALaboratory Manual, Cold Spring Harbor Laboratory Press.

In a specific embodiment, an antibody described herein is an antibody(e.g., recombinant antibody) prepared, expressed, created or isolated byany means that involves creation, e.g., via synthesis, geneticengineering of DNA sequences. In certain embodiments, such antibodycomprises sequences (e.g., DNA sequences or amino acid sequences) thatdo not naturally exist within the antibody germline repertoire of ananimal or mammal (e.g., human) in vivo.

In a certain aspect, provided herein is a method of making an antibodyor an antigen-binding fragment thereof which immunospecifically binds toGITR (e.g., human GITR) comprising culturing a cell or host celldescribed herein. In a certain aspect, provided herein is a method ofmaking an antibody or an antigen-binding fragment thereof whichimmunospecifically binds to GITR (e.g., human GITR) comprisingexpressing (e.g., recombinantly expressing) the antibody orantigen-binding fragment thereof using a cell or host cell describedherein (e.g., a cell or a host cell comprising polynucleotides encodingan antibody described herein). In a particular embodiment, the cell isan isolated cell. In a particular embodiment, the exogenouspolynucleotides have been introduced into the cell. In a particularembodiment, the method further comprises the step of purifying theantibody or antigen-binding fragment thereof obtained from the cell orhost cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York).

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow E & Lane D,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. For example, monoclonal antibodies can be producedrecombinantly from host cells exogenously expressing an antibodydescribed herein or a fragment thereof, for example, light chain and/orheavy chain of such antibody.

In specific embodiments, a “monoclonal antibody,” as used herein, is anantibody produced by a single cell (e.g., hybridoma or host cellproducing a recombinant antibody), wherein the antibodyimmunospecifically binds to GITR (e.g., human GITR) as determined, e.g.,by ELISA or other antigen-binding or competitive binding assay known inthe art or in the Examples provided herein. In particular embodiments, amonoclonal antibody can be a chimeric antibody or a humanized antibody.In certain embodiments, a monoclonal antibody is a monovalent antibodyor multivalent (e.g., bivalent) antibody. In particular embodiments, amonoclonal antibody is a monospecific or multispecific antibody (e.g.,bispecific antibody). Monoclonal antibodies described herein can, forexample, be made by the hybridoma method as described in Kohler G &Milstein C (1975) Nature 256: 495 or can, e.g., be isolated from phagelibraries using the techniques as described herein, for example. Othermethods for the preparation of clonal cell lines and of monoclonalantibodies expressed thereby are well known in the art (see, forexample, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5thEd., Ausubel F M et al., supra).

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. For example,in the hybridoma method, a mouse or other appropriate host animal, suchas a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunizedto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein (e.g., GITR (e.g.,human GITR)) used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Lymphocytes then are fused with myeloma cells usinga suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding J W (Ed), Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986)). Additionally, a RIMMS(repetitive immunization multiple sites) technique can be used toimmunize an animal (Kilpatrick K E et al., (1997) Hybridoma 16:381-9,incorporated by reference in its entirety).

In some embodiments, mice (or other animals, such as rats, monkeys,donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen(e.g., GITR (e.g., human GITR)) and once an immune response is detected,e.g., antibodies specific for the antigen are detected in the mouseserum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theAmerican Type Culture Collection (ATCC®) (Manassas, Va.), to formhybridomas. Hybridomas are selected and cloned by limited dilution. Incertain embodiments, lymph nodes of the immunized mice are harvested andfused with NS0 myeloma cells.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asNS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif., USA, and SP-2 or X63-Ag8.653 cells available from the AmericanType Culture Collection, Rockville, Md., USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor D (1984) J Immunol133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against GITR (e.g., humanGITR). The binding specificity of monoclonal antibodies produced byhybridoma cells is determined by methods known in the art, for example,immunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding J W (Ed), Monoclonal Antibodies: Principles and Practice,supra). Suitable culture media for this purpose include, for example,D-MEM or RPMI 1640 medium. In addition, the hybridoma cells may be grownin vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Antibodies described herein include antibody fragments which recognizespecific GITR (e.g., human GITR) and can be generated by any techniqueknown to those of skill in the art. For example, Fab and F(ab′)₂fragments described herein can be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)₂ fragments). A Fab fragmentcorresponds to one of the two identical arms of an antibody molecule andcontains the complete light chain paired with the VH and CH1 domains ofthe heavy chain. A F(ab′)₂ fragment contains the two antigen-bindingarms of an antibody molecule linked by disulfide bonds in the hingeregion.

Further, the antibodies described herein or antigen-binding fragmentsthereof can also be generated using various phage display methods knownin the art. In phage display methods, functional antibody domains aredisplayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In particular, DNA sequencesencoding VH and VL domains are amplified from animal cDNA libraries(e.g., human or murine cDNA libraries of affected tissues). The DNAencoding the VH and VL domains are recombined together with a scFvlinker by PCR and cloned into a phagemid vector. The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13, and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to a particular antigen can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Examples of phage display methodsthat can be used to make the antibodies described herein include thosedisclosed in Brinkman U et al., (1995) J Immunol Methods 182: 41-50;Ames R S et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene187: 9-18; Burton D R & Barbas C F (1994) Advan Immunol 57: 191-280; PCTApplication No. PCT/GB91/001134; International Publication Nos. WO90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO95/15982, WO 95/20401, and WO 97/13844; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceantibody fragments such as Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax R L et al., (1992) BioTechniques12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; andBetter M et al., (1988) Science 240: 1041-1043.

In one aspect, to generate whole antibodies, PCR primers including VH orVL nucleotide sequences, a restriction site, and a flanking sequence toprotect the restriction site can be used to amplify the VH or VLsequences from a template, e.g., scFv clones. Utilizing cloningtechniques known to those of skill in the art, the PCR amplified VHdomains can be cloned into vectors expressing a VH constant region, andthe PCR amplified VL domains can be cloned into vectors expressing a VLconstant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₂, IgG₃ and IgG₄. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 andEP 519596; Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G Met al., (1994) Prot Engineering 7(6): 805-814; and Roguska M A et al.,(1994) PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No.5,766,886, International Publication No. WO 93/17105; Tan P et al.,(2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng.13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M etal., (1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996)Protein Eng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23Supp): 5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22;Sandhu J S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) JMol Biol 235(3): 959-73. See also U.S. Application Publication No. US2005/0042664 A1 (Feb. 24, 2005), which is incorporated by referenceherein in its entirety.

Methods for making multispecific (e.g., bispecific antibodies) have beendescribed, see, for example, U.S. Pat. Nos. 7,951,917; 7,183,076;8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well known in the art. See RiechmannL & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al.,(2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) JBiotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591 and WO 01/44301.

Further, antibodies that immunospecifically bind to a GITR antigen can,in turn, be utilized to generate anti-idiotype antibodies that “mimic”an antigen using techniques well known to those skilled in the art.(See, e.g., Greenspan N S & Bona C A (1989) FASEB J 7(5): 437-444; andNissinoff A (1991) J Immunol 147(8): 2429-2438).

In particular embodiments, an antibody described herein, which binds tothe same epitope of GITR (e.g., human GITR) as an anti-GITR antibodydescribed herein, is a human antibody or an antigen-binding fragmentthereof. In particular embodiments, an antibody described herein, whichcompetitively blocks (e.g., in a dose-dependent manner) any one of theantibodies described herein, (e.g., Hum231#1, Hum231#2, pab1964,pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972,pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, pab2161,or Hum231#2w) from binding to GITR (e.g., human GITR), is a humanantibody or an antigen-binding fragment thereof. Human antibodies can beproduced using any method known in the art. For example, transgenic micewhich are incapable of expressing functional endogenous immunoglobulins,but which can express human immunoglobulin genes, can be used. Inparticular, the human heavy and light chain immunoglobulin genecomplexes can be introduced randomly or by homologous recombination intomouse embryonic stem cells. Alternatively, the human variable region,constant region, and diversity region can be introduced into mouseembryonic stem cells in addition to the human heavy and light chaingenes. The mouse heavy and light chain immunoglobulin genes can berendered non-functional separately or simultaneously with theintroduction of human immunoglobulin loci by homologous recombination.In particular, homozygous deletion of the J_(H) region preventsendogenous antibody production. The modified embryonic stem cells areexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of anantigen (e.g., GITR). Monoclonal antibodies directed against the antigencan be obtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg N & Huszar D (1995) Int RevImmunol 13:65-93. For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., International Publication Nos.WO 98/24893, WO 96/34096 and WO 96/33735; and U.S. Pat. Nos. 5,413,923,5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and5,939,598. Examples of mice capable of producing human antibodiesinclude the Xenomouse™ (Abgenix, Inc.; U.S. Pat. Nos. 6,075,181 and6,150,184), the HuAb-Mouse™ (Mederex, Inc./Gen Pharm; U.S. Pat. Nos.5,545,806 and 5,569,825), the Trans Chromo Mouse™ (Kirin) and the KMMouse™ (Medarex/Kirin).

Human antibodies which specifically bind to GITR (e.g., human GITR) canbe made by a variety of methods known in the art including phage displaymethods described above using antibody libraries derived from humanimmunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887, 4,716,111,and 5,885,793; and International Publication Nos. WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741.

In some embodiments, human antibodies can be produced using mouse-humanhybridomas. For example, human peripheral blood lymphocytes transformedwith Epstein-Barr virus (EBV) can be fused with mouse myeloma cells toproduce mouse-human hybridomas secreting human monoclonal antibodies,and these mouse-human hybridomas can be screened to determine ones whichsecrete human monoclonal antibodies that immunospecifically bind to atarget antigen (e.g., GITR (e.g., human GITR)). Such methods are knownand are described in the art, see, e.g., Shinmoto H et al., (2004)Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31.

5.2.1 Polynucleotides

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody described herein or a fragmentthereof (e.g., a variable light chain region and/or variable heavy chainregion) that immunospecifically binds to a GITR (e.g., human GITR)antigen, and vectors, e.g., vectors comprising such polynucleotides forrecombinant expression in host cells (e.g., E. coli and mammaliancells). Provided herein are polynucleotides comprising nucleotidesequences encoding any of the antibodies provided herein, as well asvectors comprising such polynucleotide sequences, e.g., expressionvectors for their efficient expression in host cells, e.g., mammaliancells.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular lessthan about 10%) of other material, e.g., cellular material, culturemedium, other nucleic acid molecules, chemical precursors and/or otherchemicals. In a specific embodiment, a nucleic acid molecule(s) encodingan antibody described herein is isolated or purified.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies or antigen-binding fragmentsthereof, which immunospecifically bind to a GITR polypeptide (e.g.,human GITR) and comprises an amino acid sequence as described herein, aswell as antibodies which compete with such antibodies for binding to aGITR polypeptide (e.g., in a dose-dependent manner), or which binds tothe same epitope as that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody described herein. The polynucleotides can comprise nucleotidesequences encoding a light chain comprising the VL FRs and CDRs ofantibodies described herein (see, e.g., Tables 1 and 3). Thepolynucleotides can comprise nucleotide sequences encoding a heavy chaincomprising the VH FRs and CDRs of antibodies described herein (see,e.g., Tables 2 and 4). In specific embodiments, a polynucleotidedescribed herein encodes a VL domain comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 202, 204, 205, 207,208, and 400-518. In specific embodiments, a polynucleotide describedherein encodes a VL domain comprising the amino acid sequence of SEQ IDNO: 519. In specific embodiments, a polynucleotide described hereinencodes a VH domain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 201, 203, 206, and 215-389. In specificembodiments, a polynucleotide described herein encodes a VL domaincomprising the amino acid sequence of any one of antibodies 231-32-15,Hum231#1 or Hum231#2 (e.g., SEQ ID NOs: 202, 207 or 208). In specificembodiments, a polynucleotide described herein encodes a VH domaincomprising the amino acid sequence of any one of antibodies 231-32-15,Hum231#1 or Hum231#2 (e.g., SEQ ID NOs: 201 or 206). In specificembodiments, a polynucleotide described herein encodes a VL domain and aVH domain comprising the amino acid sequence of any one of antibodies231-32-15, Hum231#1 or Hum231#2 (e.g., SEQ ID NOs: 201-202 and/or206-208).

In particular embodiments, provided herein are polynucleotidescomprising a nucleotide sequence encoding an anti-GITR antibodycomprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, andVL CDR3 of any one of antibodies described herein (e.g., see Table 1,for example, the VL CDRs in one row in Table 1). In specificembodiments, provided herein are polynucleotides comprising three VHchain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of any one ofantibodies described herein (e.g., see Table 2, for example, the VH CDRsin one row in Table 2). In specific embodiments, provided herein arepolynucleotides comprising a nucleotide sequence encoding an anti-GITRantibody comprising three VH chain CDRs, e.g., containing VL CDR1, VLCDR2, and VL CDR3 of any one of antibodies described herein (e.g., seeTable 1, e.g., the VL CDRs in one row in Table 1) and three VH chainCDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of any one ofantibodies described herein (e.g., see Table 2, e.g., the VH CDRs in onerow in Table 2). In specific embodiments, a polynucleotide describedherein encodes the VL CDRs of any one of antibodies 231-32-15, Hum231#1or Hum231#2 (e.g., SEQ ID NOs: 16, 17, or 18). In specific embodiments,a polynucleotide described herein encodes the VH CDRs of any one ofantibodies 231-32-15, Hum231#1 or Hum231#2 (e.g., SEQ ID NOs: 13, 14, or15). In specific embodiments, a polynucleotide described herein encodesVL CDRs and VH CDRs of any one of antibodies 231-32-15, Hum231#1 orHum231#2 (e.g., SEQ ID NOs: 13-18).

In particular embodiments, provided herein are polynucleotidescomprising a nucleotide sequence encoding an anti-GITR antibodycomprising a VL domain, e.g., containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4,comprising an amino acid sequence described herein (e.g., see Tables 1and 3, e.g., the VL CDRs and VLFRs of a particular antibody identifiedby name in the tables). In specific embodiments, provided herein arepolynucleotides comprising a nucleotide sequence encoding an anti-GITRantibody comprising a VH domain, e.g., containingFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, comprising an amino acid sequencedescribed herein (e.g., see Tables 2 and 4, e.g., the VH CDRs and VH FRsof a particular antibody identified by name in the Tables).

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an antibody provided herein comprising alight chain variable region comprising an amino acid sequence describedherein (e.g., SEQ ID NOs: 202, 204, 205, 207, 208, and 400-518 or SEQ IDNO:519), wherein the antibody immunospecifically binds to GITR (e.g.,human GITR). In a certain embodiment, a polynucleotide described hereincomprises a nucleotide sequence encoding antibodies Hum231#1 or Hum231#2or Hum231#2w provided herein comprising a light chain variable regioncomprising an amino acid sequence described herein (e.g., SEQ ID NOs:207 or 208).

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an antibody provided herein comprising aheavy chain variable region comprising an amino acid sequence describedherein (e.g., SEQ ID NO: 201, 203, 206, and 215-389), wherein theantibody immunospecifically binds to GITR (e.g., human GITR). In acertain embodiment, a polynucleotide described herein comprises anucleotide sequence encoding antibodies Hum231#1, Hum231#2 or Hum231#2wprovided herein comprising a heavy chain variable region comprising anamino acid sequence described herein (e.g., SEQ ID NO: 206).

In certain aspects, a polynucleotide comprises a nucleotide sequenceencoding an antibody described herein comprising a VL domain comprisingone or more VL FRs having the amino acid sequence described herein(e.g., see Table 3, e.g., the framework regions in one row of thetable), wherein the antibody immunospecifically binds to GITR (e.g.,human GITR). In certain aspects, a polynucleotide comprises a nucleotidesequence encoding an antibody described herein comprising a VH domaincomprising one or more VH FRs having the amino acid sequence describedherein (e.g., see Table 4, e.g., the framework regions in one row of thetable), wherein the antibody immunospecifically binds to GITR (e.g.,human GITR).

In specific embodiments, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein comprising:framework regions (e.g., framework regions of the VL domain and VHdomain) that are human framework regions, wherein the antibodyimmunospecifically binds GITR (e.g., human GITR). In certainembodiments, a polynucleotide provided herein comprises a nucleotidesequence encoding an antibody or fragment thereof (e.g., CDRs orvariable domain) described in Section 5.1 above.

In specific aspects, provided herein is a polynucleotide comprising anucleotide sequence encoding an antibody comprising a light chain and aheavy chain, e.g., a separate light chain and heavy chain. With respectto the light chain, in a specific embodiment, a polynucleotide providedherein comprises a nucleotide sequence encoding a kappa light chain. Inanother specific embodiment, a polynucleotide provided herein comprisesa nucleotide sequence encoding a lambda light chain. In yet anotherspecific embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein comprising ahuman kappa light chain or a human lambda light chain. In a particularembodiment, a polynucleotide provided herein comprises a nucleotidesequence encoding an antibody described herein, which immunospecificallybinds to GITR (e.g., human GITR), wherein the antibody comprises a lightchain, and wherein the amino acid sequence of the VL domain can compriseany amino acid sequence described herein (e.g., SEQ ID NO: 202, 204,205, 207, 208 and 400-518 or SEQ ID NO:519), and wherein the constantregion of the light chain comprises the amino acid sequence of a humankappa light chain constant region. In another particular embodiment, apolynucleotide provided herein comprises a nucleotide sequence encodingan antibody described herein, which immunospecifically binds to GITR(e.g., human GITR), and comprises a light chain, wherein the amino acidsequence of the VL domain can comprises any amino acid sequencedescribed herein (e.g., SEQ ID NO: 202, 204, 205, 207, 208 and 400-518or SEQ ID NO:519), and wherein the constant region of the light chaincomprises the amino acid sequence of a human lambda light chain constantregion. For example, human constant region sequences can be thosedescribed in U.S. Pat. No. 5,693,780.

In a particular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), wherein theantibody comprises a heavy chain, wherein the amino acid sequence of theVH domain can comprise any amino acid sequence described herein (e.g.,SEQ ID NO: 201, 203, 206 and 215-389), and wherein the constant regionof the heavy chain comprises the amino acid sequence of a human gamma(γ) heavy chain constant region.

In a certain embodiment, a polynucleotide provided herein comprises anucleotide sequence(s) encoding a VH domain and/or a VL domain of anantibody described herein (e.g., Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107 such as SEQ ID NO: 209 or 800-974 for theVH domain or SEQ ID NO: 210, 211 or 1001-1126 for the VL domain), whichimmunospecifically binds to GITR (e.g., human GITR). In a certainembodiment, a polynucleotide provided herein comprises a nucleotidesequence(s) encoding a VH domain and/or a VL domain of an antibodydescribed herein (e.g., Hum231#1, Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15, orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161 such as SEQID NO: 209 or 800-974 for the VH domain or SEQ ID NO: 210, 211 or1000-1118 for the VL domain), which immunospecifically binds to GITR(e.g., human GITR). In a certain embodiment, a polynucleotide providedherein comprises a nucleotide sequence(s) encoding a VH domain and/or aVL domain of antibody Hum231#1 or Hum231# (e.g., SEQ ID NOs: 209-211).

In yet another specific embodiment, a polynucleotide provided hereincomprises a nucleotide sequence encoding an antibody described herein(or an antigen-binding fragment thereof), which immunospecifically bindsGITR (e.g., human GITR), wherein the antibody comprises a VL domain anda VH domain comprising any amino acid sequences described herein, andwherein the constant regions comprise the amino acid sequences of theconstant regions of a human IgG₁ (e.g., allotype 1, 17, or 3) or humanIgG₄.

In a specific embodiment, provided herein are polynucleotides comprisinga nucleotide sequence encoding an anti-GITR antibody, or anantigen-binding fragment or domain thereof, designated herein, see,e.g., Tables 1-4, for example antibody Hum231#1, Hum231#2, pab1964,pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972,pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983,231-32-15, or antibodies 1-107, or antibodies pab2159, pab2160, pab2161,or Hum231#2w.

Also provided herein are polynucleotides encoding an anti-GITR antibodyor a fragment thereof that are optimized, e.g., by codon/RNAoptimization, replacement with heterologous signal sequences, andelimination of mRNA instability elements. Methods to generate optimizednucleic acids encoding an anti-GITR antibody or a fragment thereof(e.g., light chain, heavy chain, VH domain, or VL domain) forrecombinant expression by introducing codon changes and/or eliminatinginhibitory regions in the mRNA can be carried out by adapting theoptimization methods described in, e.g., U.S. Pat. Nos. 5,965,726;6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly. Forexample, potential splice sites and instability elements (e.g., A/T orA/U rich elements) within the RNA can be mutated without altering theamino acids encoded by the nucleic acid sequences to increase stabilityof the RNA for recombinant expression. The alterations utilize thedegeneracy of the genetic code, e.g., using an alternative codon for anidentical amino acid. In some embodiments, it can be desirable to alterone or more codons to encode a conservative mutation, e.g., a similaramino acid with similar chemical structure and properties and/orfunction as the original amino acid. Such methods can increaseexpression of an anti-GITR antibody or fragment thereof by at least 1fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or morerelative to the expression of an anti-GITR antibody encoded bypolynucleotides that have not been optimized.

In certain embodiments, an optimized polynucleotide sequence encoding ananti-GITR antibody described herein or a fragment thereof (e.g., VLdomain and/or VH domain) can hybridize to an antisense (e.g.,complementary) polynucleotide of an unoptimized polynucleotide sequenceencoding an anti-GITR antibody described herein or a fragment thereof(e.g., VL domain and/or VH domain). In specific embodiments, anoptimized nucleotide sequence encoding an anti-GITR antibody describedherein or a fragment hybridizes under high stringency conditions toantisense polynucleotide of an unoptimized polynucleotide sequenceencoding an anti-GITR antibody described herein or a fragment thereof.In a specific embodiment, an optimized nucleotide sequence encoding ananti-GITR antibody described herein or a fragment thereof hybridizesunder high stringency, intermediate or lower stringency hybridizationconditions to an antisense polynucleotide of an unoptimized nucleotidesequence encoding an anti-GITR antibody described herein or a fragmentthereof. Information regarding hybridization conditions has beendescribed, see, e.g., U.S. Patent Application Publication No. US2005/0048549 (e.g., paragraphs 72-73), which is incorporated herein byreference.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies described herein, e.g., antibodiesdescribed in Tables 1-4, and modified versions of these antibodies canbe determined using methods well known in the art, i.e., nucleotidecodons known to encode particular amino acids are assembled in such away to generate a nucleic acid that encodes the antibody. Such apolynucleotide encoding the antibody can be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier G et al.,(1994), BioTechniques 17: 242-6), which, briefly, involves the synthesisof overlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain and/or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody described herein) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR can then be cloned into replicable cloning vectorsusing any method well known in the art.

DNA encoding anti-GITR antibodies described herein can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the anti-GITR antibodies).Hybridoma cells can serve as a source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of anti-GITR antibodies in the recombinant hostcells.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. In certain embodiments, the vectors for expressing the VH or VLdomains comprise an EF-1α promoter, a secretion signal, a cloning sitefor the variable domain, constant domains, and a selection marker suchas neomycin. The VH and VL domains can also be cloned into one vectorexpressing the necessary constant regions. The heavy chain conversionvectors and light chain conversion vectors are then co-transfected intocell lines to generate stable or transient cell lines that expressfull-length antibodies, e.g., IgG, using techniques known to those ofskill in the art.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein. In specificembodiments, polynucleotides described herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH domain (e.g., SEQ ID NO: 201, 203, 206,and 215-389) and/or VL domain (e.g., 202, 204, 205, 207, 208, and400-518 or SEQ ID NO: 519) provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3.

5.2.2 Cells and Vectors

In certain aspects, provided herein are cells (e.g., host cells)expressing (e.g., recombinantly) antibodies described herein (or anantigen-binding fragment thereof) which specifically bind to GITR (e.g.,human GITR) and related polynucleotides and expression vectors. Providedherein are vectors (e.g., expression vectors) comprising polynucleotidescomprising nucleotide sequences encoding anti-GITR antibodies or afragment for recombinant expression in host cells, preferably inmammalian cells. Also provided herein are host cells comprising suchvectors for recombinantly expressing anti-GITR antibodies describedherein (e.g., human or humanized antibody). In a particular aspect,provided herein are methods for producing an antibody described herein,comprising expressing such antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., afull-length antibody, heavy and/or light chain of an antibody, or asingle chain antibody described herein) that specifically binds to GITR(e.g., human GITR) involves construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule, heavy and/or light chainof an antibody, or a fragment thereof (e.g., heavy and/or light chainvariable domains) described herein has been obtained, the vector for theproduction of the antibody molecule can be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody or antibody fragment (e.g., light chain or heavy chain)encoding nucleotide sequence are described herein. Methods which arewell known to those skilled in the art can be used to constructexpression vectors containing antibody or antibody fragment (e.g., lightchain or heavy chain) coding sequences and appropriate transcriptionaland translational control signals. These methods include, for example,in vitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Also provided are replicable vectors comprising anucleotide sequence encoding an antibody molecule described herein, aheavy or light chain of an antibody, a heavy or light chain variabledomain of an antibody or a fragment thereof, or a heavy or light chainCDR, operably linked to a promoter. Such vectors can, for example,include the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., International Publication Nos. WO 86/05807and WO 89/01036; and U.S. Pat. No. 5,122,464) and variable domains ofthe antibody can be cloned into such a vector for expression of theentire heavy, the entire light chain, or both the entire heavy and lightchains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody described herein (e.g.,an antibody comprising the CDRs of any one of antibodies Hum231#1,Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, 231-32-15 or antibodies 1-107, or antibodies pab2159,pab2160, or pab2161) or a fragment thereof. Thus, provided herein arehost cells containing a polynucleotide encoding an antibody describedherein or fragments thereof, or a heavy or light chain thereof, orfragment thereof, or a single chain antibody described herein (e.g., anantibody comprising the CDRs of any one of antibodies Hum231#1,Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, 231-32-15 or antibodies 1-107, or antibodies pab2159,pab2160, or pab2161), operably linked to a promoter for expression ofsuch sequences in the host cell. In certain embodiments, for theexpression of double-chained antibodies, vectors encoding both the heavyand light chains, individually, can be co-expressed in the host cell forexpression of the entire immunoglobulin molecule, as detailed below. Incertain embodiments, a host cell contains a vector comprising apolynucleotide encoding both the heavy chain and light chain of anantibody described herein (e.g., an antibody comprising the CDRs of anyone of antibodies Hum231#1, Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15 orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161), or afragment thereof. In specific embodiments, a host cell contains twodifferent vectors, a first vector comprising a polynucleotide encoding aheavy chain or a heavy chain variable region of an antibody describedherein (e.g., an antibody comprising the CDRs of any one of antibodiesHum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161), or a fragment thereof, and asecond vector comprising a polynucleotide encoding a light chain or alight chain variable region of an antibody described herein (e.g., anantibody comprising the CDRs of any one of antibodies Hum231#1,Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, 231-32-15 or antibodies 1-107, or antibodies pab2159,pab2160, or pab2161), or a fragment thereof. In other embodiments, afirst host cell comprises a first vector comprising a polynucleotideencoding a heavy chain or a heavy chain variable region of an antibodydescribed herein (e.g., an antibody comprising the CDRs of any one ofantibodies Hum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967,pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976,pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies1-107, or antibodies pab2159, pab2160, or pab2161), or a fragmentthereof, and a second host cell comprises a second vector comprising apolynucleotide encoding a light chain or a light chain variable regionof an antibody described herein (e.g., an antibody comprising the CDRsof any one of antibodies Hum231#1, Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15 orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161). Inspecific embodiments, a heavy chain/heavy chain variable regionexpressed by a first cell associated with a light chain/light chainvariable region of a second cell to form an anti-GITR antibody describedherein (e.g., antibody comprising the CDRs of any one of antibodiesHum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161) or an antigen-binding fragmentthereof. In certain embodiments, provided herein is a population of hostcells comprising such first host cell and such second host cell.

In a particular embodiment, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-GITR antibody describedherein (e.g., antibody comprising the CDRs of any one of antibodiesHum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161), and a second vector comprisinga polynucleotide encoding a heavy chain/heavy chain variable region ofan anti-GITR antibody described herein (e.g., antibody comprising theCDRs of any one of antibodies Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15or antibodies 1-107, or antibodies pab2159, pab2160, or pab2161).

A variety of host-expression vector systems can be utilized to expressantibody molecules described herein (e.g., an antibody comprising theCDRs of any one of antibodies Hum231#1, Hum231#2, pab1964, pab1965,pab1966, pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973,pab1975, pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15or antibodies 1-107, or antibodies pab2159, pab2160, or pab2161 (see,e.g., U.S. Pat. No. 5,807,715). Such host-expression systems representvehicles by which the coding sequences of interest can be produced andsubsequently purified, but also represent cells which can, whentransformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule described herein in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems (e.g., green algae such as Chlamydomonasreinhardtii) infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing antibody coding sequences; or mammalian cell systems(e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6,VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210,R1.1, B-W, L-M, BSC1, BSC40, YB/20 and BMT10 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). In a specific embodiment, cells for expressingantibodies described herein (e.g., an antibody comprising the CDRs ofany one of antibodies Hum231#1, Hum231#2, pab1964, pab1965, pab1966,pab1967, pab1968, pab1969, pab1970, pab1971, pab1972, pab1973, pab1975,pab1976, pab1977, pab1979, pab1980, pab1981, pab1983, 231-32-15 orantibodies 1-107, or antibodies pab2159, pab2160, or pab2161) or anantigen-binding fragment thereof are CHO cells, for example CHO cellsfrom the CHO GS System™ (Lonza). In a particular embodiment, cells forexpressing antibodies described herein are human cells, e.g., human celllines. In a specific embodiment, a mammalian expression vector ispOptiVEC™ or pcDNA3.3. In a particular embodiment, bacterial cells suchas Escherichia coli, or eukaryotic cells (e.g., mammalian cells),especially for the expression of whole recombinant antibody molecule,are used for the expression of a recombinant antibody molecule. Forexample, mammalian cells such as Chinese hamster ovary (CHO) cells, inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies (Foecking M K & Hofstetter H (1986) Gene 45:101-5; and Cockett M I et al., (1990) Biotechnology 8(7): 662-7). Incertain embodiments, antibodies described herein are produced by CHOcells or NS0 cells. In a specific embodiment, the expression ofnucleotide sequences encoding antibodies described herein whichimmunospecifically bind GITR (e.g., human GITR) is regulated by aconstitutive promoter, inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983)EMBO J 2: 1791-1794), in which the antibody coding sequence can beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye S &Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster S M(1989) J Biol Chem 24: 5503-5509); and the like. For example, pGEXvectors can also be used to express foreign polypeptides as fusionproteins with glutathione 5-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to matrix glutathione agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV), for example, can be used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The antibody codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region Elor E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan J &Shenk T (1984) PNAS 81(12): 3655-9). Specific initiation signals canalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression can be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol.153: 516-544).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst,HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 andHsS78Bst cells. In certain embodiments, anti-GITR antibodies describedherein (e.g., an antibody comprising the CDRs of any one of antibodiesHum231#1, Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, 231-32-15 or antibodies 1-107, orantibodies pab2159, pab2160, or pab2161) are produced in mammaliancells, such as CHO cells.

In a specific embodiment, the antibodies described herein orantigen-binding fragments thereof have reduced fucose content or nofucose content. Such antibodies can be produced using techniques knownone skilled in the art. For example, the antibodies can be expressed incells deficient or lacking the ability of to fucosylate. In a specificexample, cell lines with a knockout of both alleles ofα1,6-fucosyltransferase can be used to produce antibodies orantigen-binding fragments thereof with reduced fucose content. ThePotelligent® system (Lonza) is an example of such a system that can beused to produce antibodies or antigen-binding fragments thereof withreduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an anti-GITR antibody described herein (e.g., an antibodycomprising the CDRs of any one of antibodies Hum231#1, Hum231#2,pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970, pab1971,pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980, pab1981,pab1983, 231-32-15 or antibodies 1-107, or antibodies pab2159, pab2160,or pab2161) or an antigen-binding fragment thereof can be engineered. Inspecific embodiments, a cell provided herein stably expresses a lightchain/light chain variable domain and a heavy chain/heavy chain variabledomain which associate to form an antibody described herein (e.g., anantibody comprising the CDRs of any one of antibodies Hum231#1,Hum231#2, pab1964, pab1965, pab1966, pab1967, pab1968, pab1969, pab1970,pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979, pab1980,pab1981, pab1983, 231-32-15 or antibodies 1-107, or antibodies pab2159,pab2160, or pab2161) or an antigen-binding fragment thereof.

In certain aspects, rather than using expression vectors which containviral origins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA/polynucleotide, engineered cells can be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express an anti-GITR antibodydescribed herein or a fragment thereof. Such engineered cell lines canbe particularly useful in screening and evaluation of compositions thatinteract directly or indirectly with the antibody molecule.

A number of selection systems can be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (SzybalskaE H & Szybalski W (1962) PNAS 48(12): 2026-2034) and adeninephosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23)genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (WiglerM et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance tothe aminoglycoside G-418 (Wu G Y & Wu C H (1991) Biotherapy 3: 87-95;Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C(1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) AnnRev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) TrendsBiotechnol 11(5): 211-5); and hygro, which confers resistance tohygromycin (Santerre R F et al., (1984) Gene 30(1-3): 147-56). Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone and such methods aredescribed, for example, in Ausubel F M et al., (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990);and in Chapters 12 and 13, Dracopoli N C et al., (eds.), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colbère-Garapin F et al., (1981) J Mol Biol 150: 1-14, which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington C R & Hentschel C C G, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse G F et al., (1983) Mol Cell Biol3: 257-66).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides. Thehost cells can be co-transfected with different amounts of the two ormore expression vectors. For example, host cells can be transfected withany one of the following ratios of a first expression vector and asecond expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot N J (1986) Nature322: 562-565; and Köhler G (1980) PNAS 77: 2197-2199). The codingsequences for the heavy and light chains can comprise cDNA or genomicDNA. The expression vector can be monocistronic or multicistronic. Amulticistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9,10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotidesequences. For example, a bicistronic nucleic acid construct cancomprise in the following order a promoter, a first gene (e.g., heavychain of an antibody described herein), and a second gene and (e.g.,light chain of an antibody described herein). In such an expressionvector, the transcription of both genes can be driven by the promoter,whereas the translation of the mRNA from the first gene can be by acap-dependent scanning mechanism and the translation of the mRNA fromthe second gene can be by a cap-independent mechanism, e.g., by an IRES.

Once an antibody molecule described herein has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an antibody or an antigen-binding fragmentthereof described herein is isolated or purified. Generally, an isolatedantibody is one that is substantially free of other antibodies withdifferent antigenic specificities than the isolated antibody. Forexample, in a particular embodiment, a preparation of an antibodydescribed herein is substantially free of cellular material and/orchemical precursors. The language “substantially free of cellularmaterial” includes preparations of an antibody in which the antibody isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, an antibody that issubstantially free of cellular material includes preparations ofantibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1%(by dry weight) of heterologous protein (also referred to herein as a“contaminating protein”) and/or variants of an antibody, for example,different post-translational modified forms of an antibody or otherdifferent versions of an antibody (e.g., antibody fragments). When theantibody is recombinantly produced, it is also generally substantiallyfree of culture medium, i.e., culture medium represents less than about20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the proteinpreparation. When the antibody is produced by chemical synthesis, it isgenerally substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. Accordingly, suchpreparations of the antibody have less than about 30%, 20%, 10%, or 5%(by dry weight) of chemical precursors or compounds other than theantibody of interest. In a specific embodiment, antibodies describedherein are isolated or purified.

5.3 Pharmaceutical Compositions

Provided herein are compositions comprising an antibody orantigen-binding fragment thereof described herein having the desireddegree of purity in a physiologically acceptable carrier, excipient orstabilizer (Remington's Pharmaceutical Sciences (1990) Mack PublishingCo., Easton, Pa.). Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In a specific embodiment, pharmaceutical compositions comprise anantibody or antigen-binding fragment thereof described herein, andoptionally one or more additional prophylactic or therapeutic agents, ina pharmaceutically acceptable carrier. In a specific embodiment,pharmaceutical compositions comprise an effective amount of an antibodyor antigen-binding fragment thereof described herein, and optionally oneor more additional prophylactic of therapeutic agents, in apharmaceutically acceptable carrier. See Section 5.4, infra, forexamples of prophylactic or therapeutic agents. In some embodiments, theantibody is the only active ingredient included in the pharmaceuticalcomposition. Pharmaceutical compositions described herein can be usefulin enhancing, inducing or activating a GITR activity and treating acondition, such as cancer and an infectious disease.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parenteral. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody includesterile solutions ready for injection, sterile dry soluble products,such as lyophilized powders, ready to be combined with a solvent justprior to use, including hypodermic tablets, sterile suspensions readyfor injection, sterile dry insoluble products ready to be combined witha vehicle just prior to use and sterile emulsions. The solutions may beeither aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising an antibody are prepared as described forthe local and systemic administration. The resulting mixture can be asolution, suspension, emulsions or the like and can be formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

An antibody or antigen-binding fragment thereof described herein can beformulated as an aerosol for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209 and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatment ofinflammatory diseases, particularly asthma). These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflations,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, in one embodiment, havediameters of less than 50 microns, in one embodiment less than 10microns.

An antibody or antigen-binding fragment thereof described herein can beformulated for local or topical application, such as for topicalapplication to the skin and mucous membranes, such as in the eye, in theform of gels, creams, and lotions and for application to the eye or forintracisternal or intraspinal application. Topical administration iscontemplated for transdermal delivery and also for administration to theeyes or mucosa, or for inhalation therapies. Nasal solutions of theantibody alone or in combination with other pharmaceutically acceptableexcipients can also be administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody. For example, such patches are disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

In certain embodiments, a pharmaceutical composition comprising anantibody or antigen-binding fragment thereof described herein is alyophilized powder, which can be reconstituted for administration assolutions, emulsions and other mixtures. It may also be reconstitutedand formulated as solids or gels. The lyophilized powder is prepared bydissolving an antibody or antigen-binding fragment thereof describedherein, or a pharmaceutically acceptable derivative thereof, in asuitable solvent. In some embodiments, the lyophilized powder issterile. The solvent may contain an excipient which improves thestability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

The antibodies or antigen-binding fragments thereof described herein andother compositions provided herein can also be formulated to be targetedto a particular tissue, receptor, or other area of the body of thesubject to be treated. Many such targeting methods are well known tothose of skill in the art. All such targeting methods are contemplatedherein for use in the instant compositions. For non-limiting examples oftargeting methods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552,6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495,6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366,5,900,252, 5,840,674, 5,759,542 and 5,709,874. In a specific embodiment,an antibody or antigen-binding fragment thereof described herein istargeted to a tumor.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

5.4 Uses and Methods 5.4.1 Therapeutic Uses and Methods

In one aspect, presented herein are methods for modulating one or moreimmune functions or responses in a subject, comprising to a subject inneed thereof administering an anti-GITR antibody or antigen-bindingfragment thereof described herein, or a composition thereof. In aspecific aspect, presented herein are methods for activating, enhancingor inducing one or more immune functions or responses in a subject,comprising to a subject in need thereof administering an anti-GITRantibody or antigen-binding fragment thereof, or a composition thereof.In a specific embodiment, presented herein are methods for preventingand/or treating diseases in which it is desirable to activate or enhanceone or more immune functions or responses, comprising administering to asubject in need thereof an anti-GITR antibody or antigen-bindingfragment thereof described herein or a composition thereof. In otherspecific embodiments, the method comprises combination therapy, whereinthe anti-GITR antibody or antigen-binding fragment thereof isadministered to a subject in combination with another therapy, such asthose described below, to activate or enhance one or more immunefunctions or responses. In certain embodiments, the anti-GITR antibodyor antigen-binding fragment thereof is administered as an adjuvant incombination with an antigenic composition. In certain embodiments, theantigenic composition comprises a cancer or tumor antigen (e.g., thebcr/abl antigen in leukemia, HPVE6 and E7 antigens of the oncogenicvirus associated with cervical cancer, the MAGE1 and MZ2-E antigens inor associated with melanoma, or the MVC-1 and HER-2 antigens in orassociated with breast cancer). In some embodiments, the antigeniccomposition comprises an antigen derived from a pathogen (e.g., a viralantigen, parasitic antigen, bacterial antigen or fungal antigen).Examples of viral antigens include the nucleoprotein (NP) of influenzavirus, HIV antigens (e.g., gag proteins of HIV, HIV env protein (e.g.,gp120 and/or gp41), HIV Nef protein, HIV Pol proteins, HIV reversetranscriptase, or HIV protease), Ebola virus (EBOV) antigens (e.g., EBOVNP or glycoprotein), small pox antigens, hepatitis A, B or C virusantigens, human rhinovirus antigens, Herpes simplex virus antigens,poliovirus antigens, foot-and-mouth disease virus (FMDV) antigens,rabies virus antigens, rotavirus antigens, coxsackie virus antigens, andhuman papilloma virus (HPV) antigens. Examples of bacterial antigensinclude Bordetella pertussis (e.g., P69 protein and filamentoushaemagglutinin (FHA) antigens), Vibrio cholerae, Bacillus anthracis, andE. coli antigens such as E. coli heat Labile toxin B subunit (LT-B), E.coli K88 antigens, and enterotoxigenic E. coli antigens.

As used herein, the term “in combination” refers to the use of more thanone therapy (e.g., one or more prophylactic and/or therapeutic agents).The use of the term “in combination” does not restrict the order inwhich therapies are administered to a subject with a disease ordisorder, or the route of administration. A first therapy (e.g., aprophylactic or therapeutic agent) can be administered prior to (e.g., 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapy (e.g., a prophylactic or therapeutic agent) to a subjectwith a disease or disorder or a symptom thereof. In certain embodiments,a therapy (e.g., an agent) administered in combination with an anti-GITRantibody or antigen-binding fragment thereof to a subject isadministered in the same composition (e.g., pharmaceutical composition).In other embodiments, a therapy (e.g., an agent) administered incombination with an anti-GITR antibody or antigen-binding fragmentthereof is administered to a subject in a different composition (e.g.,two or more pharmaceutical compositions). The two compositions may beadministered at the same or different times and/or by the same ordifferent routes of administration. In a particular embodiment, ananti-GITR antibody or antigen-binding fragment thereof described hereinis administered to a subject in combination with a vaccine compositionto induce, activate or enhance the immune response elicited by thevaccine composition. In one embodiment, the vaccine composition is acancer vaccine. A cancer vaccine is an agent, molecule, or immunogenwhich stimulates or elicits an endogenous immune response in anindividual or subject against one or more cancer antigens. The cancerantigen can be a tumor associated peptide, or protein that induces orenhances immune response and is derived from tumor associated genes andencoded proteins including, for example, MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGEXp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), tyrosinase, brainglycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1,SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7,alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27,cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusionprotein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11,hsp70-2, KIAAO205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9,pm1-RARα fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras,GnTV, Herv-K-mel, Lage-1, Mage-C2, NA-88, /Lage-2, SP17, and TRP2-Int2,(MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3, p15(58), CEA,NY-ESO (LAGE), SCP-1, Hom/Mel-40, p53, H-Ras, HER-2/neu, BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA,human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p16, TAGE,PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, .alpha.-fetoprotein,13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242,CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175,M344, MA-50, MG7-Ag, MOV18, NB\170K, NYCO-1, RCAS1, SDCCAG16, TA-90(Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72,TLP, and TPS. Cancer vaccines are useful to either increase recognitionof cancer cells by the immune system or enhance the anti-tumor responsethrough lymphocyte activation. Effector T cells have been successfullygenerated by immunization with intact tumor cells or extract, purifiedantigens, use of peptides optimized for binding to both MHC and TcR,immune dominant peptides, DNA encoding tumor antigens, recombinantviruses encoding tumor antigens or antigen pulsed antigen-presentingcells. In some embodiments, enhancement of immune recognition and cellexpansion may be improved by the use of co-stimulators and cytokines,injection of vectors to express cytokines, in vitro antigen-pulsed andactivated autologous dendritic cells and by blocking negative modulators(e.g., using immune checkpoint targeting agents) and by depletingT-regulatory cells.

In a specific embodiment, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with a heat shock protein based tumor vaccine or a heatshock protein based pathogen vaccine. See Sections 5.4.1.1 and 5.4.1.2below regarding heat shock protein based tumor vaccines or heat shockprotein based pathogen vaccines for use in combination with an anti-GITRantibody or antigen-binding fragment thereof described herein.

In a particular embodiment, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered in combination with anadjuvant to induce, activate or enhance the agonistic effects of theanti-GITR antibody. Various adjuvants can be used depending on thetreatment context. Non-limiting examples of appropriate adjuvantsinclude, for example, Complete Freund's Adjuvant (CFA), IncompleteFreund's Adjuvant (IFA), motanide ISA (incomplete seppic adjuvant), theRibi adjuvant system (RAS), Titer Max, muramyl peptides, Syntex AdjuvantFormulation (SAF), alum (aluminum hydroxide and/or aluminum phosphate),aluminum salt adjuvants, Gerbu® adjuvants, nitrocellulose absorbedantigen, encapsulated or entrapped antigen, immuno-stimulating complexessuch as saponins, Quil A, QS-21 and others. Other adjuvants include CpGoligonucleotides and double stranded RNA molecules, such as poly(A),poly(U). Combinations of the above adjuvants may also be used. In someembodiments, one or more adjuvants are a saponin, such as QS-21, QS-21and 3 De-O-acylated monophosphoryl lipid A (3 D-MPL), andimmunostimulatory oligonucleotides and saponin adjuvants as disclosed inU.S. Pat. Nos. 6,645,495; 7,029,678 and 7,858,589, respectively.

In certain embodiments, provided herein are methods for enhancing thestimulation of GITR-responsive cells (e.g., T cells, such as effectorT-cells) comprising incubating ex vivo the GITR-responsive cells (e.g.,T cells) with an antibody or antigen-binding fragment thereof describedherein. In some embodiments, the GITR-responsive cells are incubatedwith a stimulating agent (e.g., phytohaemagglutinin (PHA) and/or phorbolmyristate acetate (PMA), or a TCR complex stimulating antibody, such asan anti-CD3 antibody and anti-CD28 antibody) prior to, simultaneouslywith or subsequent to the incubation with the anti-GITR antibody orantigen-binding fragment thereof. In certain embodiments, theGITR-responsive cells (e.g., T cells) were isolated from a subject(e.g., a human). In some embodiments, the GITR-responsive cellsfollowing stimulation with the anti-GITR antibody or antigen bindingfragment thereof are administered to a subject (e.g., a human). TheGITR-response cells (e.g., T cells) may be administered to the same or adifferent subject than the cells were originally isolated from.

In some embodiments, provided herein are methods for activatingGITR-responsive cells (e.g., T cells) comprising incubating theGITR-responsive cells (e.g., T cells) with an antibody orantigen-binding fragment thereof described herein. In certainembodiments, the GITR-responsive cells are incubated with a stimulatingagent (e.g., a T cell receptor complex stimulating agent such as, e.g.,phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or aTCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody) prior to, simultaneously with or subsequent to theincubation with the anti-GITR antibody or antigen-binding fragmentthereof. In some embodiments, the GITR-responsive cells (e.g., T cells)were isolated from a subject (e.g., a human). In certain embodiments,the GITR-responsive cells following activation with the anti-GITRantibody or antigen-binding fragment thereof are administered to asubject (e.g., a human). The GITR-responsive cells (e.g., T cells) maybe administered to the same or a different subject than the cells wereoriginally isolated from.

In some embodiments, cells responsive to GITR (i.e., GITR-responsivecells) are incubated in cell culture with an anti-GITR antibody orantigen-binding fragment thereof described herein, and administered to asubject to enhance immune function (e.g., to enhance theexpansion/proliferation of GITR-responsive cells, such as T cells,and/or enhance T cell effector function) and/or to treat cancer and/orprevent or treat an infectious disease. Examples of cancers andinfectious diseases are provided herein. See, e.g., Example 7 below forexemplary methods. In specific embodiments, the GITR-responsive cellsare effector T cells (e.g., CD4⁺ and CD8⁺). In some embodiments, theGITR-responsive cells are isolated from a subject. In some embodiments,the GITR-responsive cells are assessed for GITR expression prior toincubation with an anti-GITR antibody or antigen-binding fragmentthereof described herein. In certain embodiments, the GITR-responsivecells are incubated with a mitogen (e.g., a T cell mitogen, such as,e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA),or a TCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody) prior to, simultaneously with or subsequent to theincubation with an anti-GITR antibody or antigen-binding fragmentthereof described herein. The GITR responsive cells may be incubatedwith an anti-GITR antibody or antigen-binding fragment thereof describedherein for, e.g., 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours,12 hours, 18 hours, 24 hours or more. In certain embodiments, theGITR-responsive cells which are administered to a subject were derivedfrom the subject (i.e., the GITR-responsive cells are autologous). Inother embodiments, the GITR-responsive cells which are administered to asubject were derived from a different subject. The GITR-responsive cellsfollowing incubation with anti-GITR antibody or antigen-binding fragmentthereof may be administered locally or systemically to a subject via anyroute known to one of skill in the art (e.g., parenteral administration,such as subcutaneous, intravenous, or intramuscular administration, orintratumoral administration). In certain embodiments, a suitable dose ofGITR-responsive cells following incubation with anti-GITR antibody or anantigen-binding fragment thereof administered to subject may be at least100, 200, 300, 400, 500, 700, 1,000, 5,000, 10,000, 25,000, 50,000,100,000, 1×10⁶, 1×10⁷, or 1×10⁸ cells. The GITR-responsive cellsfollowing incubation with anti-GITR antibody or antigen-binding fragmentthereof may be administered 1, 2, 3, 4, 5, 6, 7, 8 or more times. Thefrequency and dose of GITR-responsive cells following incubation withanti-GITR antibody or antigen-binding fragment thereof which areadministered to a subject will vary depending on several factors,including, e.g., the condition of the patient. In another embodiment,provided herein is a method for enhancing the expansion of T cells(e.g., CD4⁺ and/or CD8⁺ T cells) in a subject, comprising administeringto the subject an effective amount of an antibody or antigen-bindingfragment thereof described herein, or a pharmaceutical compositiondescribed herein. In another embodiment, provided herein is a method forenhancing the expansion of CD8⁺ T cells in a subject, comprisingadministering to the subject an effective amount of an antibody orantigen-binding fragment thereof described herein, or a pharmaceuticalcomposition described herein. In another embodiment, provided herein isa method for enhancing the expansion of CD4⁺ T cells in a subject,comprising administering to the subject an effective amount of anantibody or antigen-binding fragment thereof described herein, or apharmaceutical composition described herein. In a specific embodiment,the subject is human.

In another embodiment, provided herein is a method for enhancing theexpansion of T cells (e.g., CD4⁺ and/or CD8⁺ T cells) and/or T celleffector function in a subject, comprising administering to the subjectan effective amount of an antibody or antigen-binding fragment thereofdescribed herein, or a pharmaceutical composition described herein. Inanother embodiment, provided herein is a method for enhancing theexpansion of CD8⁺ T cells and/or T cell effector function in a subject,comprising administering to the subject an effective amount of anantibody or antigen-binding fragment thereof described herein, or apharmaceutical composition described herein. In another embodiment,provided herein is a method for enhancing the expansion of CD4+ T cellsand/or T cell effector function in a subject, comprising administeringto the subject an effective amount of an antibody or antigen-bindingfragment thereof described herein, or a pharmaceutical compositiondescribed herein. In a specific embodiment, the subject is human.

In another embodiment, provided herein is a method for preferentialexpansion of effector T-cells over the expansion of T-regulatory cells,comprising incubating ex vivo T-cells with an antibody orantigen-binding fragment thereof described herein. In certainembodiments, the anti-GITR antibody or antigen-binding fragment thereofexpands effector T-cells over T-regulatory cells by 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or greater. Insome embodiments, the anti-GITR antibody or antigen-binding fragmentthereof expands effector T-cells over T-regulatory cells by 10% to 20%,15% to 25%, 25% to 50%, 30% to 60%, 50% to 75% or 65% to 85%. Theeffector T-cells and T-regulatory cells can be distinguished from eachother by cell surface markers, such as those disclosed in the examplesinfra. In some embodiments, the T-cells were isolated from a subject(e.g., a human). In certain embodiments, the T-cells after expansion areadministered to a subject (e.g., a human).

In another embodiment, provided herein is a method for preferentialexpansion of effector T-cells over the expansion of T-regulatory cellsin a subject, comprising administering to the subject an effectiveamount of an antibody or antigen-binding fragment thereof describedherein, or a composition thereof. In certain embodiments, the anti-GITRantibody or antigen-binding fragment thereof expands effector T-cellsover T-regulatory cells by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80% or greater. In some embodiments, theanti-GITR antibody or antigen-binding fragment thereof expands effectorT-cells over T-regulatory cells by 10% to 20%, 15% to 25%, 25% to 50%,30% to 60%, 50% to 75% or 65% to 85%. The effector T-cells andT-regulatory cells can be distinguished from each other by cell surfacemarkers and/or intracellular markers, such as those disclosed in theexamples infra. In a specific embodiment, the subject is human.

In certain embodiments, treatment of a subject with an anti-GITRantibody or antigen-binding fragment described herein, or a compositionthereof achieves one, two, three, four or more of the following effects:(i) reduction or amelioration of the severity of a disease or symptomassociated therewith; (ii) reduction in the duration of a symptomassociated with a disease; (iii) inhibition of the progression of adisease or symptom associated therewith; (iv) regression of a disease orsymptom associated therewith; (v) prevention of the development or onsetof a symptom associated with a disease that is present in the patient;(vi) inhibition of the recurrence of a symptom associated with adisease; (vii) reduction in the hospitalization of a subject; (viii)reduction in the hospitalization length; (ix) an increase in thesurvival of a subject with a disease; (x) a reduction in the number ofsymptoms associated with a disease; and (xi) an enhancement,improvement, supplementation, complementation or augmentation of thetherapeutic effect(s) of another therapy. In an alternative embodiment,an anti-GITR antibody or antigen-binding fragment thereof is used toprevent a disorder, such as an infectious disease.

In some embodiments, an anti-GITR antibody or antigen-binding fragmentthereof, or a composition thereof is administered to a subject incombination with an immunotherapeutic agent. Immunotherapeutic agentsfor use in the combination therapies disclosed herein include, but arenot limited to, a Her2/neu receptor antibody such as trastuzumab(marketed as Herceptin®), an anti-CD52 antibody such as alemtuzumab(marketed as Campath®. MabCampath® or Campath-1H), an anti-CD33 antibodysuch as gemtuzumab linked to calicheamicin (marketed as Mylotarg®), ananti-CD20 antibody such as rituximab (marketed as Rituxan® andMabThera®), Ibritumomab tiuxetan (marketed as Zevalin®), anti-TNFαantibodies such as infliximab (marketed as Remicade®) or adalimmumab(marketed as Humira®), a soluble TNFR2 molecule such as etanercept(marketed as Enbrel®), an antibody to the CD25 chain of the IL-2receptor such as basiliximab (marketed as Simulect®), an anti-CD40/CD40Lantibody such as humanized IgG₁ anti-human CD40 antibody (SGN-40),Toll-like receptor agonists such as monophosphoril lipid A (MPL®), CpG,single-stranded RNA, nucleotides, nucleotide analogue, CL087 (aTLR7-specific ligand), loxoribine, polyinosinepolycytidylic acid,flagellin, resiquimod, immiquimod, gardiquimod, NOD ligands such asmuramyl dipeptide, murabutide, peptidoglycan and muramyldipeptide, CD1dagonists, such as α-galactosyl ceramide (α-GalCer) and threitol ceramide(ThrCer), an antibody such as Fresolimumab® (GC1008), an antibodytargeting an inhibiting TGF-beta isoforms 1, 2 or 3, an Fc fusion,Dalantercept (Alk-Fc), and the small molecule LY2157299 (receptor kinaseinhibitor).

Diseases that can be treated by an enhancement of immune functioninclude cancer and infectious diseases. Various cancers and infectiousdiseases are described below. In a specific embodiment, an anti-GITRantibody or antigen-binding fragment thereof described herein can beused to treat a condition associated with cancer or a conditionresulting from the administration of an anti-cancer therapy (such as,e.g., chemotherapy or radiation). In a particular embodiment, ananti-GITR antibody or antigen-binding fragment thereof can be used totreat or manage lymphocytopenia. In another embodiment, an anti-GITRantibody or antigen-binding fragment thereof is administered to apatient diagnosed with cancer to increase the proliferation and/oreffector function of one or more immune cell populations (e.g., T celleffector cells, such as CD4⁺ and CD8⁺ T cells) in the patient.

In a specific embodiment, an anti-GITR antibody or antigen-bindingfragment thereof described herein activates or enhances or induces oneor more immune functions or responses in a subject by at least 99%, atleast 98%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10%, or in the range of between 10% to 25%, 25%to 50%, 50% to 75%, or 75% to 95% relative to the immune function in asubject not administered the anti-GITR antibody or antigen-bindingfragment thereof described herein using assays well known in the art,e.g., ELISPOT, ELISA, and cell proliferation assays. In a specificembodiment, the immune function is cytokine production (e.g.,interferon-gamma, IL-2, IL-5, IL-10, IL-12, or transforming growthfactor (TGF)-alpha production). In another embodiment, the immunefunction is T cell proliferation/expansion, which can be assayed, e.g.,by flow cytometry to detect the number of cells expressing markers of Tcells (e.g., CD3, CD4, or CD8). In another embodiment, the immunefunction is antibody production, which can be assayed, e.g., by ELISA.In some embodiments, the immune function is effector function, which canbe assayed, e.g., by a cytotoxicity assay or other assays well known inthe art. In another embodiment, the immune function is a Th1 response.In another embodiment, the immune function is a Th2 response. In anotherembodiment, the immune function is a memory response.

In specific embodiments, non-limiting examples of immune functions thatmay be enhanced or induced by an anti-GITR antibody or antigen-bindingfragment thereof are proliferation/expansion of effector lymphocytes(e.g., increase in the number of effector T lymphocytes), inhibition ofapoptosis of effector lymphocytes (e.g., effector T lymphocytes), andsuppression of Tregs. In particular embodiments, an immune functionenhanced or induced by an anti-GITR antibody or antigen-binding fragmentthereof described herein is proliferation/expansion in the number of oractivation of CD4⁺ T cells (e.g., Th1 and Th2 helper T cells), CD8⁺ Tcells (e.g., cytotoxic T lymphocytes, alpha/beta T cells, andgamma/delta T cells), B cells (e.g., plasma cells), memory T cells,memory B cells, tumor-resident T cells, CD122⁺ T cells, natural killer(NK) cells), macrophages, monocytes, dendritic cells, mast cells,eosinophils, basophils or polymorphonucleated leukocytes. In oneembodiment, an anti-GITR antibody or antigen-binding fragment thereofdescribed herein activates or enhances the proliferation/expansion ornumber of lymphocyte progenitors. In some embodiments, an anti-GITRantibody or antigen-binding fragment thereof described herein increasesthe number of CD4⁺ T cells (e.g., Th1 and Th2 helper T cells), CD8⁺ Tcells (e.g., cytotoxic T lymphocytes, alpha/beta T cells, andgamma/delta T cells), B cells (e.g., plasma cells), memory T cells,memory B cells, tumor-resident T cells, CD122⁺ T cells, natural killercells (NK cells), macrophages, monocytes, dendritic cells, mast cells,eosinophils, basophils or polymorphonucleated leukocytes byapproximately at least 99%, at least 98%, at least 95%, at least 90%, atleast 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10%, or in the rangeof between 10% to 25%, 25% to 50%, 50% to 75%, or 75% to 95% relative anegative control (e.g., number of the respective cells not treated,cultured, or contacted with an anti-GITR antibody or antigen-bindingfragment thereof described herein).

In particular embodiments, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), induces, activatesor enhances an activity of human GITR independent of TCR triggering. Inspecific embodiments, an antibody described herein, whichimmunospecifically binds to GITR (e.g., human GITR), induces, activatesor enhances an activity of NF-κB independent of TCR triggering. Incertain embodiments, the activity of NF-κB can be assessed in, e.g., anassay comprising the following steps: (a) incubating T cells (e.g.,Jurkat cells) expressing a NF-κB-luciferase reporter construct (e.g.,GloResponse NF-κB-luc2P construct) and GITR (e.g., human GITR) with theantibody described herein or an isotype control antibody at an antibodyconcentration of, e.g., 12.5, 10, 5, 2.5, 1.25, or 0.625 μg/ml, in theabsence of an anti-CD3 antibody; and (b) reading luciferase signalafter, e.g., 2, 5, 6, 8 or 18 hours of incubation using, e.g., anEnVision multilabel reader 2100, wherein a positive luciferase signalrelative to the isotype control antibody indicates the activity ofNF-κB. In a particular embodiment, the luciferase signal is read after 5hours of incubation.

In another embodiment, provided herein is a method of activating T cellsindependent of TCR triggering comprising contacting T cells with anantibody or antigen-binding fragment thereof described herein. Inparticular embodiments, provided herein is a method of inducing,activating or enhancing an activity of NF-κB independent of TCRtriggering comprising contacting T cells with an antibody orantigen-binding fragment thereof described herein. In certainembodiments, the activity of NF-κB can be assessed in, e.g., an assaycomprising the following steps: (a) incubating T cells (e.g., Jurkatcells) expressing a NF-κB-luciferase reporter construct (e.g.,GloResponse NF-κB-luc2P construct) and GITR (e.g., human GITR) with theantibody described herein or an isotype control antibody at an antibodyconcentration of, e.g., 12.5, 10, 5, 2.5, 1.25, or 0.625 μg/ml, in theabsence of an anti-CD3 antibody; and (b) reading luciferase signalafter, e.g., 2, 5, 6, 8 or 18 hours of incubation using, e.g., anEnVision multilabel reader 2100, wherein a positive luciferase signalrelative to the isotype control antibody indicates the activity ofNF-κB. In a particular embodiment, the luciferase signal is read after 5hours of incubation.

In particular embodiments, provided herein is a method of increasing thepercentage of polyfunctional (IFNγ+ TNFα+) T cells comprising contactingT cells with an antibody or antigen-binding fragment thereof describedherein. An increase in the percentage of polyfunctional (IFNγ+ TNFα+) Tcells can be assessed in, e.g., an assay comprising the following steps:(a) incubating, e.g., human PBMCs with, e.g., an anti-CD3 antibody atvarious suboptimal concentrations (e.g., 0.3-5 μg/ml); and, e.g., anantibody described herein, which immunospecifically binds to GITR (e.g.,human GITR), at, e.g., 5 μg/ml or an isotype control antibody, for,e.g., 3-4 days at 37° C. and 5% CO₂; (b) treating cells with, e.g.,Brefeldin A for, e.g., 6 hours at 37° C. and 5% CO₂; (c) staining thesurface of the cells using, e.g., an anti-CD3 antibody, an anti-CD4antibody, and an anti-CD8α antibody; (d) staining intracellularly using,e.g., an anti-IFNγ antibody and an anti-TNFα antibody; and (e)determining the percentage of polyfunctional (IFNγ+ TNFα+) T cellsrelative to the isotype control antibody. In specific embodiments, thepolyfunctional (IFNγ+ TNFα+) T cells are selected from the groupconsisting of polyfunctional (IFNγ+ TNFα+) CD4+ T cells andpolyfunctional (IFNγ+ TNFα+) CD8+ T cells.

In particular embodiments, provided herein is a method of increasingsurface expression of OX40 and PD-1 in activated T cells comprisingcontacting T cells with an antibody or antigen-binding fragment thereofdescribed herein. The surface expression of OX40 and PD-1 in activated Tcells can be increased by at least about 1.2 fold, 1.3 fold, 1.4 fold,1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold,6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold,300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, or1000 fold as assessed by methods described herein and/or known to one ofskill in the art, relative to surface expression of OX40 and PD-1 inactivated T cells without the antibody described herein.

5.4.1.1 Cancer

In a specific aspect, presented herein are methods for treating cancer,comprising administering to a subject in need thereof an effectiveamount of an anti-GITR antibody or antigen-binding fragment thereofdescribed herein or a composition thereof. In a specific embodiment, ananti-GITR antibody or antigen-binding fragment thereof described hereinor a composition thereof is the only active agent administered to asubject.

The effect of an anti-GITR antibody or antigen-binding fragment thereofdescribed herein on proliferation of cancer cells can be detected byroutine assays, such as by assays that measure the uptake ofradiolabeled thymidine. Alternatively, cell viability can be measured byassays that measure lactate dehydrogenase (LDH), a stable cytosolicenzyme that is released upon cell lysis, or by the release of [⁵¹Cr]upon cell lysis. In one embodiment, necrosis measured by the ability orinability of a cell to take up a dye such as neutral red, trypan blue,or ALAMAR™ blue (Page B et al., (1993) Intl J Oncology 3: 473-6). Insuch an assay, the cells are incubated in media containing the dye, thecells are washed, and the remaining dye, reflecting cellular uptake ofthe dye, is measured spectrophotometrically.

In another embodiment, the dye is sulforhodamine B (SRB), whose bindingto proteins can be used as a measure of cytotoxicity (Skehan P et al.,(1990) J Nat Cancer Inst 82: 1107-12). In yet another embodiment, atetrazolium salt, such as MTT, is used in a quantitative colorimetricassay for mammalian cell survival and proliferation by detecting living,but not dead, cells (see, e.g., Mosmann T (1983) J Immunol Methods 65:55-63).

In other embodiments, apoptotic cells are measured in both the attachedand “floating” compartments of the cultures. Both compartments arecollected by removing the supernatant, trypsinizing the attached cells,and combining both preparations following a centrifugation wash step (10minutes, 2000 rpm). The protocol for treating tumor cell cultures withsulindac and related compounds to obtain a significant amount ofapoptosis has been described in the literature (see, e.g., Piazza G A etal., (1995) Cancer Res 55: 3110-6). Features of this method includecollecting both floating and attached cells, identification of theoptimal treatment times and dose range for observing apoptosis, andidentification of optimal cell culture conditions.

In another embodiment, apoptosis is quantitated by measuring DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, (1999) 2: 34 37 (Roche Molecular Biochemicals). In yetanother embodiment, apoptosis can be observed morphologically.

Cancer cell lines on which such assays can be performed are well knownto those of skill in the art. Apoptosis, necrosis and proliferationassays can also be performed on primary cells, e.g., a tissue explant.

In specific embodiments, the administration of an anti-GITR antibody orantigen-binding fragment thereof described herein or a compositionthereof to a subject with cancer (in some embodiments, an animal modelfor cancer) achieves at least one, two, three, four or more of thefollowing effects: (i) the reduction or amelioration of the severity ofone or more symptoms of cancer; (ii) the reduction in the duration ofone or more symptoms associated with cancer; (iii) the prevention in therecurrence of a symptom associated with cancer; (iv) the reduction inhospitalization of a subject; (v) a reduction in hospitalization length;(vi) the increase in the survival of a subject; (vii) the enhancement orimprovement of the therapeutic effect of another therapy; (viii) theinhibition of the development or onset of one or more symptomsassociated with cancer; (ix) the reduction in the number of symptomsassociated with cancer; (x) improvement in quality of life as assessedby methods well known in the art; (x) inhibition of the recurrence of atumor; (xi) the regression of tumors and/or one or more symptomsassociated therewith; (xii) the inhibition of the progression of tumorsand/or one or more symptoms associated therewith; (xiii) a reduction inthe growth of a tumor; (xiv) a decrease in tumor size (e.g., volume ordiameter); (xv) a reduction in the formation of a newly formed tumor;(xvi) eradication, removal, or control of primary, regional and/ormetastatic tumors; (xvii) a decrease in the number or size ofmetastases; (xviii) a reduction in mortality; (xix) an increase inrelapse free survival; (xx) the size of the tumor is maintained and doesnot increase or increases by less than the increase of a tumor afteradministration of a standard therapy as measured by conventional methodsavailable to one of skill in the art, such as magnetic resonance imaging(MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray, and computedtomography (CT) scan, or a positron emission tomography (PET) scan;and/or (xxi) an increase in the length of remission in patients.

In certain embodiments, two or more different anti-GITR antibodies orantigen-binding fragments thereof described herein are administered to asubject. In some embodiments, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with one or more other therapies, e.g., anti-cancer agents,cytokines, cellular vaccines or anti-hormonal agents, to treat cancer.

In a specific embodiment, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered in combination withradiation therapy comprising, e.g., the use of x-rays, gamma rays andother sources of radiation to destroy the cancer cells. In specificembodiments, the radiation treatment is administered as external beamradiation or teletherapy wherein the radiation is directed from a remotesource. In other embodiments, the radiation treatment is administered asinternal therapy or brachytherapy wherein a radioactive source is placedinside the body close to cancer cells or a tumor mass. In one aspect, ananti-GITR antibody or antigen-binding fragment thereof described hereincan activate or enhance the immune function or response in a cancerpatient with a compromised immune system due to anti-cancer therapy.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered to a subject in combinationwith chemotherapy. In an embodiment, an anti-GITR antibody orantigen-binding fragment thereof described herein can be used before,during or after radiation therapy or chemotherapy. Examples ofchemotherapeutic agents include cyclophosphamide, methotrexate,cyclosporin A, leflunomide, cisplatin, ifosfamide, taxanes such as taxoland paclitaxol, topoisomerase I inhibitors (e.g., CPT 11, topotecan, 9AC, and GG 211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil(5-FU), leucovorin, vinorelbine, temodal, cytochalasin B, gramicidin D,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin homologs, and cytoxan.

In one embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered to a subject in combinationwith cyclophosphamide, e.g., a low dose of cyclophosphamide.Cyclophosphamide (Elostan, Cytoxan), is a chemotherapeutic agent thathas an immunomodulatory function when used at low doses (e.g., up to 300mg/m², 300 mg/m² or about 300 mg/m² when administered intravenously).Specifically, low doses of cyclophosphamide can reduce the number andproliferative capacity of regulatory T cells (Treg) (e.g.,CD4+CD25+FoxP3+ cells or, alternatively, CD45+CD3+CD4+CD8−FOXP3+CD25hiCD127low cells) and modulate immune-suppressive networks. Insome embodiments, the dosage of a cyclophosphamide administration isabout 50 mg/m², 100 mg/m², 200 mg/m², 300 mg/m², 500 mg/m², or more. Insome embodiments, the dosage of a cyclophosphamide administration is ina range of 10 mg/m² to 100 mg/m², 50 mg/m² to 200 mg/m², 50 mg/m² to 300mg/m², 50 mg/m² to 500 mg/m². In some embodiments, cyclophosphamide isadministered to the subject within 1 hour, 2 hours, 3 hours, 4 hours, 8hours, 12 hours, 1 day, 5 days or more prior to or following an initialadministration of an anti-GITR antibody or antigen-binding fragmentthereof described herein. In some embodiments, anti-GITR antibody orantigen-binding fragment thereof described herein is used in combinationwith cyclophosphamide, e.g., low dose cyclophosphamide, for thetreatment of metastatic renal cell carcinoma (RCC).

In one embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered to a subject in combinationwith a Treg-inhibitory agent. Examples of Treg-inhibitory agents includeZenapax® (daclizumab) (Roche), which is a human anti-CD25 monoclonalantibody used, e.g., for inducing immune suppression in organtransplantation. Daclizumab blocks IL-2 binding to CD25, which is also asignal for the maintenance of Tregs. Another agent that inhibits Tregsis Sutent® (Sunitinib) (Pfizer) which is a small molecule,multi-targeted tyrosine kinase inhibitor approved for the treatment ofrenal cell carcinoma (RCC) and other cancers. Another agent that mayinhibit Tregs is 1-Methyl-D-tryptophan (1-MT), a competitive inhibitorof indoleamine 2,3-dioxygenase (IDO). IDO is an immunosuppressive agentexpressed in certain normal and neoplastic cells and may be associatedwith an increase in Tregs in cancer patients. Additional Treg inhibitorsinclude agents that block trafficking of Tregs to the tumormicroenvironment. Such agents may include antibodies against certainchemokines and chemokine receptors such as CCL17, CCL22 and CCR4.

Further examples of Treg-inhibitory agents that may be used inaccordance with methods described herein are disclosed in the followingpatent applications, which are incorporated herein by reference in theirentirety for all purposes: U.S. Patent Publication Nos. US 2009/0214533,US 2012/0142750, US 2011/0305713, US 2009/0004213, US 2012/0219559, US2010/0278844, US 2013/0323283 and US 2008/0152665.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein can be administered to a subject before, duringor after surgery.

In certain embodiments, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with an immune modulator or antibody. Immune modulators orantibodies may be, but are not limited to, adjuvants, antigens, anti-CD3(e.g., OKT3), checkpoint targeting agents or modulators, orinterleukins. The terms “checkpoint targeting agent” or “checkpointmodulator” can be used interchangeably and refer to an agent thatselectively modulates expression or activity of a regulatory molecule(e.g., a co-inhibitory checkpoint molecule (e.g., protein) or acostimulatory checkpoint molecule (e.g., protein), which may be, e.g., areceptor or a ligand) of an immune system checkpoint. Checkpointtargeting agents can be selected from the group consisting of an agonistof a checkpoint molecule, an antagonist of a checkpoint molecule, apolypeptide (e.g., a peptide ligand, an antibody, an antibody fragment)that selectively targets a checkpoint molecule; a small molecule thatselectively targets a checkpoint molecule; and a regulatory nucleic acid(e.g., an siRNA, miRNA) that selectively modulates expression oractivity of a checkpoint molecule. In one embodiment, a checkpointtargeting agent can be selected from the group consisting of anantagonist of PD-1, an antagonist of PD-L1, an antagonist of PD-L2, anantagonist of CTLA-4, an antagonist of TIM-3, an antagonist of LAG-3, anagonist of GITR, and an agonist of OX40. Thus, in some embodiments, ananti-GITR agonistic antibody (e.g., Hum231#1, Hum231#2 or Hum231#2w) orantigen-binding fragment thereof, may be administered in combinationwith, e.g., an anti-CTLA-4 antagonist antibody or antigen-bindingfragment thereof, or another checkpoint targeting agent, either in asingle pharmaceutical composition, or in separate pharmaceuticalcompositions administered together or separately.

In some embodiments, provided herein are methods for treating cancer ina subject comprising administering to the subject an anti-GITR antibodyor antigen-binding fragment thereof and an agonist of OX40 and/or anantagonist(s) of LAG-3, TIM-3, PD-1 and/or CTLA-4. In some embodiments,the cancer is selected from glioblastoma multiforme, metastaticmelanoma, resistant metastatic melanoma, metastatic ovarian cancer,metastatic renal cell carcinoma, head and neck cancer, gastric cancer,esophageal cancer, non-small cell lung cancer, pediatric brain tumors,low-grade asctrocytoma, ependymoma, and meduloblastoma.

In a specific embodiment, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with an agent that inhibits (partially or completely) CTLA-4signal transduction, such as an antibody that specifically binds tohuman CTLA-4 (e.g., tremelimumab (Pfizer); ipilimumab (Yervoy®,Bristol-Myers Squibb)) or a CTLA-4-Ig fusion protein. In someembodiments, an anti-GITR antibody or antigen-binding fragment thereofis used in combination with an antagonist of CTLA-4 (e.g., tremelimumabor ipilimumab) for the treatment of metastatic ovarian cancer. In someembodiments, an anti-GITR antibody or antigen-binding fragment thereofis used in combination with an antagonist of CTLA-4 (e.g., tremelimumabor ipilimumab) for the treatment of metastatic ovarian cancer that isresistant to an antagonist of CTLA-4 (e.g., tremelimumab or ipilimumab).In some embodiments, an anti-GITR antibody or antigen-binding fragmentthereof and an antagonist of CTLA-4 (e.g., tremelimumab or ipilimumab)are used for the treatment of glioblastoma multiforme. In someembodiments, the glioblastoma multiforme is recurrent. In someembodiments, the glioblastoma multiforme is newly diagnosed. In someembodiments, the glioblastoma multiforme is in a subject havingnon-methylated MGMT promoters. In some embodiments, the glioblastomamultiforme is refractory to Bevacizumab therapy. In some embodiments,the glioblastoma multiforme is in a subject that has not receivedBevacizumab therapy. Examples of anti-CTLA-4 antibodies that may be usedin treatment methods disclosed herein are disclosed in the followingpatents and patent applications, which are incorporated herein byreference in their entirety for all purposes: International PublicationNos. WO 00/037504, WO 01/014424 and WO 09/100140; U.S. Pat. Nos.6,207,156 and 7,034,121. Further examples of anti-CTLA-4 antibodies thatmay be used in accordance with the methods described herein aredisclosed in the following patents and patent applications, which areincorporated herein by reference in their entirety for all purposes,U.S. Pat. Nos. 7,465,446, 8,263,073; 8,142,778 and 8,226,946; and U.S.Application Nos. US 2003/086930, US 2005/226875, US 2007/243184, US2009/123477 and US 2011/044953.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered in combination with an agentthat inhibits (partially or completely) LAG-3 signal transduction, suchas an antibody that specifically binds to human LAG-3. Examples ofanti-LAG-3 antibodies or antibody fragments thereof that may be used intreatment methods described herein are disclosed in the followingpatents and patent applications, which are incorporated herein byreference in their entirety for all purposes: U.S. Pat. Nos. 6,143,273and 6,197,524; U.S. Patent Publication Nos. US 2011/0150892, US2010/0233183 and US 2010/196394.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered in combination with an agentthat inhibits (partially or completely) TIM-3 signal transduction, suchas an antibody that specifically binds to human TIM-3. Examples ofanti-TIM-3 antibodies or antibody fragments thereof that may be used intreatment methods described herein are disclosed in the followingpatents and patent applications, which are incorporated herein byreference in their entireties for all purposes: U.S. Pat. Nos. 7,470,428and 8,101,176; U.S. Publication Nos. US 2013/0022623, US 2010/0100131,US 2010/0100131 and US 2010/061992.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered in combination with an agentthat inhibits (partially or completely) PD-1 signal transduction, suchas an antibody that specifically binds to human PD-1. In someembodiments, an anti-PD-1 antibody or antibody fragment thereof isadministered to a subject as described herein. In some embodiments, theanti-PD-1 antibody is Nivolumab (BMS-936558 or MDX1106) or Lambrolizumab(MK-3475) or Pidilizumab (CT-011). Further non-limiting examples ofanti-PD-1 antibodies that may be used in treatment methods disclosedherein are disclosed in the following patents and patent applications,which are incorporated herein by reference in their entireties for allpurposes: U.S. Pat. Nos. 6,808,710; 7,488,802; 8,008,449; 8,114,845 and8,168,757, U.S. Publication No. US 2013/0202623 and PCT Publication No.WO 2013/033091.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered in combination with an agentthat inhibits (partially or completely) PD-L1 activity, such as anantibody that specifically binds to human PD-L1. In some embodiments,the anti-PD-L1 antibody is BMS-936559, MPDL3280A, MEDI4736 orMSB0010718C. Further non-limiting examples of anti-PD-L1 antibodies thatmay be used in treatment methods disclosed herein are disclosed in thefollowing patents and patent applications, which are incorporated hereinby reference in their entireties for all purposes: U.S. Pat. No.8,168,179 and U.S. Publication Nos. US 2010/0203056 and US 2003/0232323.In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered in combination with an agentthat inhibits (partially or completely) PD-L2 activity, such as anantibody that specifically binds to human PD-L2.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered in combination with an agentthat activates or enhances OX-40 signal transduction, such as anantibody that specifically binds to human OX-40. Examples of anti-OX40antibodies or antibody fragments thereof that may be used in treatmentmethods described herein are disclosed in the following patents andpatent applications, which are incorporated herein by reference in theirentireties for all purposes: U.S. Pat. Nos. 7,550,140; 7,807,156;8,283,450; 8,614,295 and 7,531,170; U.S. Publication No. US2010/0196359, US 2010/0136030 and US 2013/0183315.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered to a subject in combinationwith a vaccine, such as described herein, including Section 5.4.1 above.In a specific embodiment, an anti-GITR antibody or antigen-bindingfragment thereof is administered to a subject in combination with a heatshock protein based tumor-vaccine. Heat shock proteins (HSPs) are afamily of highly conserved proteins found ubiquitously across allspecies. Their expression can be powerfully induced to much higherlevels as a result of heat shock or other forms of stress, includingexposure to toxins, oxidative stress or glucose deprivation. Fivefamilies have been classified according to molecular weight: HSP-110,-90, -70, -60 and -28. HSPs deliver immunogenic peptides through thecross-presentation pathway in antigen presenting cells (APCs) such asmacrophages and dendritic cells (DCs), leading to T cell activation.HSPs function as chaperone carriers of tumor-associated antigenicpeptides forming complexes able to induce tumor-specific immunity. Uponrelease from dying tumor cells, the HSP-antigen complexes are taken upby antigen-presenting cells (APCs) wherein the antigens are processedinto peptides that bind MHC class I and class II molecules leading tothe activation of anti-tumor CD8+ and CD4+ T cells. The immunityelicited by HSP complexes derived from tumor preparations isspecifically directed against the unique antigenic peptide repertoireexpressed by the cancer of each subject.

A heat shock protein peptide complex (HSPPC) is a protein peptidecomplex consisting of a heat shock protein non-covalently complexed withantigenic peptides. HSPPCs elicit both innate and adaptive immuneresponses. In a specific embodiment, the antigenic peptide(s) displaysantigenicity for the cancer being treated. HSPPCs are efficiently seizedby APCs via membrane receptors (mainly CD91) or by binding to Toll-likereceptors. HSPPC internalization results in functional maturation of theAPCs with chemokine and cytokine production leading to activation ofnatural killer cells (NK), monocytes and Th1 and Th-2-mediated immuneresponses. In some embodiments, HSPPCs used in methods disclosed hereincomprise one or more heat shock proteins from the hsp60, hsp70, or hsp90family of stress proteins complexed with antigenic peptides. In someembodiments, HSPPCs comprise hsc70, hsp70, hsp90, hsp110, grp170, gp96,calreticulin, or combinations of two or more thereof.

In a specific embodiment, an anti-GITR antibody or antigen-bindingfragment thereof is administered to a subject in combination with a heatshock protein peptide complex (HSPPC), e.g., heat shock protein peptidecomplex-96 (HSPPC-96), to treat cancer, e.g., glioblastoma multiforme.HSPPC-96 comprises a 96 kDa heat shock protein (Hsp), gp96, complexed toantigenic peptides. HSPPC-96 is a cancer immunotherapy manufactured froma subject's tumor and contains the cancer's antigenic “fingerprint.” Insome embodiments, this fingerprint contains unique antigens that arepresent only in that particular subject's specific cancer cells andinjection of the vaccine is intended to stimulate the subject's immunesystem to recognize and attack any cells with the specific cancerfingerprint.

In some embodiments, the HSPPC, e.g., HSPPC-96, is produced from thetumor tissue of a subject. In a specific embodiment, the HSPPC (e.g.,HSPPC-96) is produced from tumor of the type of cancer or metastatisthereof being treated. In another specific embodiment, the HSPPC (e.g.,HSPPC-96) is autologous to the subject being treated. In someembodiments, the tumor tissue is non-necrotic tumor tissue. In someembodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 grams) of non-necrotic tumor tissue is used to produce avaccine regimen. In some embodiments, after surgical resection,non-necrotic tumor tissue is frozen prior to use in vaccine preparation.In some embodiments, the HSPPC, e.g., HSPPC-96, is isolated from thetumor tissue by purification techniques, filtered and prepared for aninjectable vaccine. In some embodiments, a subject is administered 6-12doses of the HSPPC, e.g., HSPCC-96. In such embodiments, the HSPPC,e.g., HSPPC-96, doses may be administered weekly for the first 4 dosesand then biweekly for the 2-8 additional doses.

In some embodiments, an anti-GITR antibody or antigen-binding fragmentthereof is used in combination with HSPPC-96 for the treatment ofmetastatic melanoma (e.g., resistant metastatic melanoma), metastaticovarian cancer, or metastatic renal cell carcinoma. In some embodiments,an anti-GITR antibody or antigen-binding fragment thereof is used incombination with HSPPC-96 for the treatment of glioblastoma multiforme.In some embodiments, an anti-GITR antibody or antigen-binding fragmentthereof and an antagonist of CTLA-4 are used in combination withHSPPC-96 for the treatment of glioblastoma multiforme. In someembodiments, the subject to be treated is immunosuppressed (e.g., due toinfection, e.g., HIV, or by reason of having undergone anti-cancertherapy (e.g., chemotherapy radiation) prior to administration of theHSPPC.

Further examples of HSPPCs that may be used in accordance with themethods described herein are disclosed in the following patents andpatent applications, which are incorporated herein by reference in theirentireties for all purposes, U.S. Pat. Nos. 6,391,306, 6,383,492,6,403,095, 6,410,026, 6,436,404, 6,447,780, 6,447,781 and 6,610,659.

In some embodiments, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered to a subject in combinationwith a compound that targets an immunomodulatory enzyme(s) such as IDO(indoleamine-(2,3)-dioxygenase) and TDO (tryptophan 2,3-dioxygenase). Inparticular embodiments, such compound is selected from the groupconsisting of epacadostat (Incyte Corporation), F001287 (FlexusBiosciences), indoximod (NewLink Genetics), and NLG919 (NewLinkGenetics). In one embodiment, the compound is epacadostat. In anotherembodiment, the compound is F001287. In another embodiment, the compoundis indoximod. In another embodiment, the compound is NLG919.

Elevated numbers of CD4⁺CD25⁺ Tregs in cancer patients prevent the hostfrom mounting an effective anti-tumor immune response. Additionally,high Treg frequencies are associated with reduced patient survival(Curiel T J et al., (2004) Nat Medicine 10(9): 942-9; Woo E Y et al.,(2002) J Immunol 168: 4272-6). However, an enhancement of immuneactivity in cancer patients' PBMC was observed after depletion ofregulatory T cells (Dannull J et al., (2005) J Clin Invest 115(12):3623-33). Tregs expressing high levels of GITR on their surface can bedepleted with anti-GITR antibody, DTA-1 (Coe D et al., (2010) CancerImmunol Immunother 59: 1367-77). In one embodiment, an immune-basedstrategy may incorporate ex-vivo or in-vivo Treg depletion with ananti-GITR antibody or antigen-binding fragment thereof causing depletionof GITR-positive Treg cells.

In certain embodiments, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with an oncolytic virus such as Talimogene laherparepvec(OncoVEX GM-CSF) and CGTG-102 (Ad5/3-D24-GMCSF).

In certain embodiments, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with a cytokine(s) that is effective in inhibiting tumorgrowth/metastasis. Such cytokines, lymphokines, or other hematopoieticfactors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF,Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.

In certain embodiments, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with a receptor tyrosine kinase inhibitor(s) such asimatinib mesylate (marketed as Gleevec® or Glivac®), erlotinib (an EGFreceptor inhibitor) now marketed as Tarveca, or sunitinib (marketed asSutent®).

In certain embodiments, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with an antagonistic TGF-beta antibody, such asFresolimumab® (GC1008), an antibody targeting and inhibiting TGF-beta 1,2 or 3 isoforms.

In some embodiments, an anti-GITR antibody or antigen-binding fragmentthereof described herein or a composition thereof is administered to asubject suffering from or diagnosed with cancer. In a specificembodiment, an anti-GITR antibody or composition thereof is administeredto a subject suffering from or diagnosed with a glioblastoma multiforme.In some embodiments, the glioblastoma multiforme is recurrent. In someembodiments, the glioblastoma multiforme is newly diagnosed. In someembodiments, the glioblastoma multiforme is in a subject havingnon-methylated MGMT promoters. In some embodiments, the glioblastomamultiforme is recurrent. In some embodiments, the glioblastomamultiforme is newly diagnosed. In some embodiments, the glioblastomamultiforme is in a subject having non-methylated MGMT promoters. In someembodiments, the glioblastoma multiforme is refractory to Bevacizumabtherapy. In some embodiments, the glioblastoma multiforme is in asubject that has not received Bevacizumab therapy.

In certain embodiments, the patients being treated in accordance withthe methods described herein are patients already being treated withantibiotics, anti-cancer agents, or other biologicaltherapy/immunotherapy. Among these patients are refractory patients,patients who are too young for conventional therapies, and patients withreoccurring viral infections despite management or treatment withexisting therapies.

In some embodiments, the subject being administered an anti-GITRantibody or antigen-binding fragment thereof described herein or acomposition thereof has not received a therapy prior to theadministration of the antibody or composition thereof. In otherembodiments, an anti-GITR antibody or antigen-binding fragment thereofdescribed herein or a composition thereof is administered to a subjectwho has received a therapy prior to administration of the antibody orcomposition thereof.

In certain embodiments, an anti-GITR antibody or antigen-bindingfragment thereof, or composition thereof is administered to a subjectreceiving or recovering from immunosuppressive therapy. In certainembodiments, an anti-GITR antibody or composition thereof isadministered to a subject with cancer and the cancer cells expressGITRL. In some embodiments, the subject to be treated in accordance withthe methods disclosed herein is immunosuppressed (e.g., due toinfection, e.g., HIV, or by reason of having undergone anti-cancertherapy (e.g., chemotherapy radiation).

As described herein, anti-GITR antibodies affect the surface expressionof proteins including OX40, CD25, and PD-1. Accordingly, in certainembodiments, an anti-GITR antibody or antigen-binding fragment thereofor composition thereof is administered prior to the administration of anantibody or antigen-binding fragment thereof that binds to OX40, CD25,or PD-1.

An antagonist PD-1 antibody or antigen-binding fragment thereof can beadministered at a time at which the agonist GITR antibody orantigen-binding fragment thereof has increased surface expression ofPD-1 in a subject relative to expression of PD-1 in the subject at thetime of the administering. For example, an antagonist PD-1 antibody orantigen-binding fragment thereof can be administered at least 12 hours,at least 1 day, at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, or at least 7 days after administrationof the agonist GITR antibody. An antagonist PD-1 antibody orantigen-binding fragment thereof can be administered from 12 hours totwo weeks, from 1 day to two weeks, from 2 days to two weeks, or from 3days to two weeks after administration of the agonist GITR antibody orantigen-binding fragment thereof. An antagonist PD-1 antibody orantigen-binding fragment thereof can be administered from 12 hours toone week, from 1 day to one week, from 2 days to one week, or from 3days to one week after administration of the agonist GITR antibody orantigen-binding fragment thereof.

An agonist OX40 antibody or antigen-binding fragment thereof can beadministered at a time at which the agonist GITR antibody orantigen-binding fragment thereof has increased surface expression ofOX40 in a subject relative to expression of OX40 in the subject at thetime of the administering. For example, an agonist OX40 antibody orantigen-binding fragment thereof can be administered at least 12 hours,at least 1 day, at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, or at least 7 days after administrationof the agonist GITR antibody. An agonist OX40 antibody orantigen-binding fragment thereof can be administered from 12 hours totwo weeks, from 1 day to two weeks, from 2 days to two weeks, or from 3days to two weeks after administration of the agonist GITR antibody orantigen-binding fragment thereof. An agonist OX40 antibody orantigen-binding fragment thereof can be administered from 12 hours toone week, from 1 day to one week, from 2 days to one week, or from 3days to one week after administration of the agonist GITR antibody orantigen-binding fragment thereof.

An anti-CD25 antibody or antigen-binding fragment thereof can beadministered at a time at which the agonist GITR antibody orantigen-binding fragment thereof has increased surface expression ofCD25 in a subject relative to expression of CD25 in the subject at thetime of the administering. For example, an anti-CD25 antibody orantigen-binding fragment thereof can be administered at least 12 hours,at least 1 day, at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, or at least 7 days after administrationof the agonist GITR antibody. An anti-CD25 antibody or antigen-bindingfragment thereof can be administered from 12 hours to two weeks, from 1day to two weeks, from 2 days to two weeks, or from 3 days to two weeksafter administration of the agonist GITR antibody or antigen-bindingfragment thereof. An anti-CD25 antibody or antigen-binding fragmentthereof can be administered from 12 hours to one week, from 1 day to oneweek, from 2 days to one week, or from 3 days to one week afteradministration of the agonist GITR antibody or antigen-binding fragmentthereof.

An antagonist CTLA-4 antibody or antigen-binding fragment thereof can beadministered after administration of an agonist GITR antibody orantigen-binding fragment. For example, an antagonist CTLA-4 antibody orantigen-binding fragment thereof can be administered at least 12 hours,at least 1 day, at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, or at least 7 days after administrationof the agonist GITR antibody. An antagonist CTLA-4 antibody orantigen-binding fragment thereof can be administered from 12 hours totwo weeks, from 1 day to two weeks, from 2 days to two weeks, or from 3days to two weeks after administration of the agonist GITR antibody orantigen-binding fragment thereof. An antagonist CTLA-4 antibody orantigen-binding fragment thereof can be administered from 12 hours toone week, from 1 day to one week, from 2 days to one week, or from 3days to one week after administration of the agonist GITR antibody orantigen-binding fragment thereof.

Examples of cancer that can be treated in accordance with the methodsdescribed herein include, but are not limited to, B cell lymphomas(e.g., B cell chronic lymphocytic leukemia, B cell non-Hodgkin lymphoma,cutaneous B cell lymphoma, diffuse large B cell lymphoma), basal cellcarcinoma, bladder cancer, blastoma, brain metastasis, breast cancer,Burkitt lymphoma, carcinoma (e.g., adenocarcinoma (e.g., of thegastroesophageal junction)), cervical cancer, colon cancer, colorectalcancer (colon cancer and rectal cancer), endometrial carcinoma,esophageal cancer, Ewing sarcoma, follicular lymphoma, gastric cancer,gastroesophageal junction carcinoma, gastrointestinal cancer,glioblastoma (e.g., glioblastoma multiforme, e.g., newly diagnosed orrecurrent), glioma, head and neck cancer (e.g., head and neck squamouscell carcinoma), hepatic metastasis, Hodgkin's and non-Hodgkin'slymphoma, kidney cancer (e.g., renal cell carcinoma and Wilms' tumors),laryngeal cancer, leukemia (e.g., chronic myelocytic leukemia, hairycell leukemia), liver cancer (e.g., hepatic carcinoma and hepatoma),lung cancer (e.g., non-small cell lung cancer and small-cell lungcancer), lymphblastic lymphoma, lymphoma, mantle cell lymphoma,metastatic brain tumor, metastatic cancer, myeloma (e.g., multiplemyeloma), neuroblastoma, ocular melanoma, oropharyngeal cancer,osteosarcoma, ovarian cancer, pancreatic cancer (e.g., pancreatis ductaladenocarcinoma), prostate cancer (e.g., hormone refractory (e.g.,castration resistant), metastatic, metastatic hormone refractory (e.g.,castration resistant, androgen independent)), renal cell carcinoma(e.g., metastatic), salivary gland carcinoma, sarcoma (e.g.,rhabdomyosarcoma), skin cancer (e.g., melanoma (e.g., metastaticmelanoma)), soft tissue sarcoma, solid tumor, squamous cell carcinoma,synovia sarcoma, testicular cancer, thyroid cancer, transitional cellcancer (urothelial cell cancer), uveal melanoma (e.g., metastatic),verrucous carcinoma, vulval cancer, and Waldenstrom macroglobulinemia.

In some embodiments, the cancer treated in accordance with the methodsdescribed herein is a human sarcoma or carcinoma, e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma (e.g., metastatic), hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, glioblastomamultiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma. In certainembodiments, the cancer treated in accordance with the methods describedherein is an acute lymphocytic leukemia or acute myelocytic leukemia(e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia); chronic leukemia (chronic myelocytic (granulocytic)leukemia or chronic lymphocytic leukemia); Hodgkin's disease;non-Hodgkin's disease; acute myeloid leukemia; B-cell lymphoma; T-celllymphoma; anaplastic large cell lymphoma; intraocular lymphoma;follicular lymphoma; small intestine lymphoma; orsplenic marginal zonelymphoma. In certain embodiments, the cancer treated in accordance withthe methods described herein is multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, gastrointestinal stromal tumors,head and/or neck cancer (e.g., squamous cell carcinoma of thehypopharynx, squamous cell carcinoma of the larynx, cell carcinoma ofthe oropharynx, or verrucous carcinoma of the larynx), endometrialstromal sarcoma, mast cell sarcoma, adult soft tissue sarcoma, uterinesarcoma, merkel cell carcinoma, urothelial carcinoma, melanoma withbrain metastases, uveal melanoma, uveal melanoma with liver metastases,non-small cell lung cancer, rectal cancer, or myelodysplastic syndrome,In some embodiments, the cancer treated in accordance with the methodsis metastatic.

In certain embodiments, the cancer treated in accordance with themethods described herein includes prostate cancer, breast cancer, lungcancer, colorectal cancer, melanoma, bronchial cancer, bladder cancer,brain or central nervous system cancer, peripheral nervous systemcancer, uterine or endometrial cancer, cancer of the oral cavity orpharynx, non-Hodgkin's lymphoma, thyroid cancer, kidney cancer, biliarytract cancer, small bowel or appendix cancer, salivary gland cancer,thyroid gland cancer, adrenal gland cancer, squamous cell cancer,mesothelioma, osteocarcinoma, thyoma/thymic carcinoma, glioblastoma,myelodysplastic syndrome, soft tissue sarcoma, DIPG, adenocarcinoma,osteosarcoma, chondrosarcoma, leukemia, or pancreatic cancer. In someembodiments, the cancer treated in accordance with the methods describedherein includes a carcinoma (e.g., an adenocarcinoma), lymphoma,blastoma, melanoma, sarcoma or leukemia. In certain embodiments, thecancer treated in accordance with the methods described herein includessquamous cell cancer, small-cell lung cancer, non-small cell lungcancer, gastrointestinal cancer, Hodgkin's lymphoma, non-Hodgkin'slymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer,ovarian cancer, liver cancer (e.g., hepatic carcinoma and hepatoma),bladder cancer, breast cancer, inflammatory breast cancer, Merkel cellcarcinoma, colon cancer, colorectal cancer, stomach cancer, urinarybladder cancer, endometrial carcinoma, myeloma (e.g., multiple myeloma),salivary gland, carcinoma, kidney cancer (e.g., renal cell carcinoma andWilms' tumors), basal cell carcinoma, melanoma, prostate cancer, vulvalcancer, thyroid cancer, testicular cancer, esophageal cancer, serousadenocarcinoma or various types of head and neck cancer. In certainembodiments, the cancer treated in accordance with the methods describedherein includes desmoplastic melanoma, inflammatory breast cancer,thymoma, rectal cancer, anal cancer, or surgically treatable ornon-surgically treatable brain stem glioma. In a specific embodiment,the cancer is a solid tumor. In another specific embodiment, the canceris glioblastoma multiforme. In some embodiments, the glioblastomamultiforme is recurrent. In some embodiments, the glioblastomamultiforme is newly diagnosed. In some embodiments, the glioblastomamultiforme is in a subject having non-methylated MGMT promoters. In someembodiments, the glioblastoma multiforme is refractory to Bevacizumabtherapy. In some embodiments, the glioblastoma multiforme is in asubject that has not received Bevacizumab therapy.

In some embodiments, the cancer treated in accordance with the methodsdescribed herein is metastatic melanoma (e.g., resistant metastaticmelanoma), metastatic ovarian cancer, or metastatic renal cellcarcinoma. In certain embodiments, the cancer treated in accordance withthe methods described herein is melanoma that is resistant toipilimumab. In some embodiments, the cancer treated in accordance withthe methods described herein is melanoma that is resistant to nivolumab.In some embodiments, the cancer treated in accordance with the methodsdescribed herein is melanoma that is resistant to ipilimumab andnivolumab.

5.4.1.2 Infectious Diseases

In a specific aspect, presented herein are methods for preventing and/ortreating an infectious disease, comprising administering to a subject inneed thereof an effective amount of an anti-GITR antibody orantigen-binding fragment thereof described herein or a compositionthereof. In one embodiment, provided herein are methods for preventingand/or treating an infection (e.g., a viral infection, a bacterialinfection, a fungal infection, a protozoal infection, or a parasiticinfection). The infection prevented and/or treated in accordance withthe methods can be caused by an infectious agent identified herein. In aspecific embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein or a composition thereof is the only activeagent administered to a subject. In some embodiments, an anti-GITRantibody or antigen-binding fragment thereof described herein or acomposition thereof is used in combination with anti-infectiveinterventions (e.g., antivirals, antibacterials, antifungals, oranti-helminthics) for the treatment of infectious diseases.

Infectious diseases that can be treated and/or prevented by an anti-GITRantibody or antigen-binding fragment thereof described herein are causedby infectious agents including but not limited to bacteria, parasites,fungi, protozae, and viruses. In a specific embodiment, the infectiousdisease treated and/or prevented by an anti-GITR antibody orantigen-binding fragment thereof described herein is caused by a virus.Viral diseases or viral infections that can be prevented and/or treatedin accordance with the methods described herein include, but are notlimited to, those caused by hepatitis type A, hepatitis type B,hepatitis type C, influenza (e.g., influenza A or influenza B),varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, small pox,Epstein Barr virus, human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), and agents of viral diseasessuch as viral meningitis, encephalitis, dengue or small pox.

Bacterial infections that can be prevented and/or treated includeinfections caused by Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseasescaused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa) that can beprevented and/or treated in accordance with the methods described hereininclude, but are not limited to, Mycobacteria rickettsia, Mycoplasma,Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillusantracis (anthrax), tetanus, Streptococcus, Staphylococcus,mycobacterium, pertissus, cholera, plague, diptheria, chlamydia, S.aureus and legionella.

Protozoal diseases or protozoal infections caused by protozoa that canbe prevented and/or treated in accordance with the methods describedherein include, but are not limited to, leishmania, coccidiosis,trypanosoma schistosoma or malaria. Parasitic diseases or parasiticinfections caused by parasites that can be prevented and/or treated inaccordance with the methods described herein include, but are notlimited to, chlamydia and rickettsia.

Fungal diseases or fungal infections that can be prevented and/ortreated in accordance with the methods described herein include, but arenot limited to, those caused by Candida infections, zygomycosis, Candidamastitis, progressive disseminated trichosporonosis with latenttrichosporonemia, disseminated candidiasis, pulmonaryparacoccidioidomycosis, pulmonary aspergillosis, Pneumocystis cariniipneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis andcerebrospinal vasculitis, Aspergillus niger infection, Fusariumkeratitis, paranasal sinus mycoses, Aspergillus fumigatus endocarditis,tibial dyschondroplasia, Candida glabrata vaginitis, oropharyngealcandidiasis, X-linked chronic granulomatous disease, tinea pedis,cutaneous candidiasis, mycotic placentitis, disseminatedtrichosporonosis, allergic bronchopulmonary aspergillosis, mycotickeratitis, Cryptococcus neoformans infection, fungal peritonitis,Curvularia geniculata infection, staphylococcal endophthalmitis,sporotrichosis, and dermatophytosis.

In certain embodiments, administering an anti-GITR antibody orantigen-binding fragment thereof described herein or a compositionthereof to a subject (in some embodiments, an animal model) achievesone, two, three, four, or more of the following effects: (i) reductionor amelioration the severity of an infectious disease, an infection orsymptom associated therewith; (ii) reduction in the duration of aninfectious disease, an infection or symptom associated therewith; (iii)inhibition of the progression of an infectious disease, an infection orsymptom associated therewith; (iv) regression of an infectious disease,an infection or symptom associated therewith; (v) inhibition of thedevelopment or onset of an infectious disease, an infection or symptomassociated therewith; (vi) inhibition of the recurrence of an infectiousdisease or symptom associated therewith; (vii) reduction or inhibitionof the spread of an infectious agent from one cell to another cell, onetissue to another tissue, or one organ to another organ; (viii)inhibition or reduction of the spread/transmission of an infectiousagent from one subject to another subject; (ix) reduction in organfailure associated with an infectious disease; (x) reduction in thehospitalization of a subject; (xi) reduction in the hospitalizationlength; (xii) an increase in the survival of a subject with aninfectious disease or an infection; (xiii) elimination of an infectiousdisease or an infection; (xiii) inhibition or reduction in replicationof an infectious agent or an infection; (xiv) inhibition or reduction inthe entry of an infectious agent into a cell(s); (xv) inhibition orreduction of replication of the genome of an infectious agent; (xvi)inhibition or reduction in the synthesis of infectious agent proteins;(xvii) inhibition or reduction in the assembly of infectious agents;(xviii) inhibition or reduction in the release of infectious agents froma cell(s); (xviii) reduction in the number or titer of an infectiousagent; (xix) the reduction in the number of symptoms associated with aninfectious disease or an infection; (xx) enhancement, improvement,supplementation, complementation, or augmentation of the prophylactic ortherapeutic effect(s) of another therapy; and/or (xxi) prevention of theonset or progression of a secondary infection associated with aninfectious disease.

In certain embodiments, two or more different anti-GITR antibodies orantigen-binding fragments thereof described herein are administered to asubject. In some embodiments, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with one or more other therapies. In one embodiment, ananti-GITR antibody or antigen-binding fragment thereof described hereinis administered in combination with one or more anti-fungals.

In a specific embodiment, an anti-GITR antibody or antigen-bindingfragment thereof described herein is administered to a subject incombination with one or more antibiotics. Examples of antibiotics, thatcan be used in combination with an anti-GITR antibody or antigen-bindingfragment thereof described herein include aminoglycoside antibiotics,glycopeptides, amphenicol antibiotics, ansamycin antibiotics,cephalosporins, cephamycins oxazolidinones, penicillins, quinolones,streptogamins, tetracyclins, and analogs thereof.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof described herein is administered in combination with one or moreanti-virals. Examples of antiviral agents that can be used incombination with an anti-GITR antibody or antigen-binding fragmentthereof described herein include non-nucleoside reverse transcriptaseinhibitors, nucleoside reverse transcriptase inhibitors, proteaseinhibitors and fusion inhibitors. In one embodiment, the antiviral agentis amantadine, oseltamivir phosphate, rimantadine, and zanamivir. Inanother embodiment, the antiviral agent is a non-nucleoside reversetranscriptase inhibitor such as delavirdine, efavirenz or nevirapine. Inanother embodiment, the antiviral agent is a nucleoside reversetranscriptase inhibitor such as abacavir, didanosine, emtricitabine,emtricitabine, lamivudine, stavudine, tenofovir DF, zalcitabine orzidovudine. In another embodiment, the antiviral agent is a proteaseinhibitor such as amprenavir, atazanavir, fosamprenav, indinavir,lopinavir, nelfinavir, ritonavir or saquinavir. In another embodiment,the antiviral agent is a fusion inhibitor such as enfuvirtide. Inanother embodiment, the antiviral agent is oseltamivir phosphate,amphotericin B or palivizumab.

In a specific embodiment, an anti-GITR antibody or antigen-bindingfragment thereof is administered to a subject in combination with avaccine that is a heat shock protein preparation comprising a heat shockprotein complexed to antigenic peptides comprising an antigen from apathogen (e.g., a virus, bacteria, fungus, etc.). In some embodiments,an anti-GITR antibody or antigen-binding fragment thereof isadministered to a subject in combination with a vaccine that is a heatshock protein preparation comprising heat shock proteins complexed withantigenic peptides comprising viral antigens (e.g., antigens of HSV-1 orHSV-2). In some embodiments, the heat shock protein preparationcomprising heat shock proteins complexed with antigenic peptidescomprising viral antigens (e.g., antigens of HSV-1 or HSV-2) is combinedwith an adjuvant, such as QS-21. In some embodiments, the vaccine isHerpV (Agenus Inc.), which is a vaccine for the treatment of herpesinfections. A non-limiting example of a suitable vaccine is disclosed inMo A., et al., (2011), Vaccine 29: 8530-8541, the contents of which areincorporated herein by reference in their entirety. Further non-limitingexamples are disclosed in U.S. Pat. No. 8,541,002, the contents of whichare incorporated herein by reference in their entirety.

In some embodiments, an anti-GITR antibody or antigen-binding fragmentthereof described herein, or composition thereof is administered to asubject suffering from an infectious disease caused by infectious agentsincluding, but not limited to bacteria, fungi, protozae and viruses. Incertain embodiments, an anti-GITR antibody or antigen-binding fragmentthereof described herein, or composition thereof is administered to asubject diagnosed as having an infectious disease caused by infectiousagents including, but not limited to bacteria, fungi, protozae andviruses. In some embodiments, an anti-GITR antibody or antigen-bindingfragment thereof described herein, or a composition thereof isadministered to a subject with an infection. In some embodiments, thesubject treated in accordance with the method described herein isimmunocompromised or immunosuppressed. In certain embodiments, thesubject treated in accordance with methods described herein has, is, orotherwise will receive another therapy (e.g., an anti-viral agent,antibiotic, or anti-fungal agent).

5.4.1.3 Routes of Administration & Dosage

An antibody or an antigen-binding fragment thereof, or compositiondescribed herein may be delivered to a subject by a variety of routes.These include, but are not limited to, parenteral, intranasal,intratracheal, oral, intradermal, topical, intramuscular,intraperitoneal, transdermal, intravenous, intratumoral, conjunctivaland subcutaneous routes. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent for use as a spray.

The amount of an antibody or an antigen-binding fragment thereof, orcomposition which will be effective in the treatment and/or preventionof a condition will depend on the nature of the disease, and can bedetermined by standard clinical techniques.

The precise dose to be employed in a composition will also depend on theroute of administration, and the seriousness of the infection or diseasecaused by it, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses may also vary depending upon means of administration, target site,physiological state of the patient (including age, body weight andhealth), whether the patient is human or an animal, other medicationsadministered, or whether treatment is prophylactic or therapeutic.Usually, the patient is a human but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages are optimallytitrated to optimize safety and efficacy.

In certain embodiments, an in vitro assay is employed to help identifyoptimal dosage ranges. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

For passive immunization with an antibody (or an antigen-bindingfragment thereof), the dosage ranges from about 0.0001 to 100 mg/kg, andmore usually 0.01 to 15 mg/kg, of the patient body weight. For example,dosages can be 1 mg/kg body weight, 10 mg/kg body weight, or within therange of 1-10 mg/kg or in other words, 70 mg or 700 mg or within therange of 70-700 mg, respectively, for a 70 kg patient. In someembodiments, the dosage administered to the patient is about 1 mg/kg toabout 20 mg/kg of the patient's body weight. Generally, human antibodieshave a longer half-life within the human body than antibodies from otherspecies due to the immune response to the foreign polypeptides. Thus,lower dosages of human antibodies and less frequent administration isoften possible.

An exemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months for a period of oneyear or over several years, or over several year-intervals. In somemethods, two or more antibodies or antigen-binding fragments thereofwith different binding specificities are administered simultaneously toa subject. An antibody or antigen-binding fragment thereof is usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly, every 3 months, every 6 months or yearly.

5.4.2 Detection & Diagnostic Uses

An anti-GITR antibody or an antigen-binding fragment thereof describedherein (see, e.g., Section 5.1) can be used to assay GITR protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art, including immunoassays, such as the enzymelinked immunosorbent assay (ELISA), immunoprecipitation, or Westernblotting. Suitable antibody assay labels are known in the art andinclude enzyme labels, such as, glucose oxidase; radioisotopes, such asiodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(¹²¹In), and technetium (⁹⁹Tc); luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin. Suchlabels can be used to label an antibody or an antigen-binding fragmentthereof described herein. Alternatively, a second antibody thatrecognizes an anti-GITR antibody or antigen-binding fragment thereofdescribed herein can be labeled and used in combination with ananti-GITR antibody or antigen-binding fragment thereof to detect GITRprotein levels.

Assaying for the expression level of GITR protein is intended to includequalitatively or quantitatively measuring or estimating the level of aGITR protein in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level) or relatively (e.g.,by comparing to the disease associated protein level in a secondbiological sample). GITR polypeptide expression level in the firstbiological sample can be measured or estimated and compared to astandard GITR protein level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” GITR polypeptide level is known, it can be used repeatedly asa standard for comparison.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing GITR. Methods for obtaining tissue biopsiesand body fluids from animals (e.g., humans) are well known in the art.Biological samples include peripheral mononuclear blood cells.

An anti-GITR antibody or antigen-binding fragment thereof describedherein can be used for prognostic, diagnostic, monitoring and screeningapplications, including in vitro and in vivo applications well known andstandard to the skilled artisan and based on the present description.Prognostic, diagnostic, monitoring and screening assays and kits for invitro assessment and evaluation of immune system status and/or immuneresponse may be utilized to predict, diagnose and monitor to evaluatepatient samples including those known to have or suspected of having animmune system-dysfunction or with regard to an anticipated or desiredimmune system response, antigen response or vaccine response. Theassessment and evaluation of immune system status and/or immune responseis also useful in determining the suitability of a patient for aclinical trial of a drug or for the administration of a particularchemotherapeutic agent or an antibody or antigen-binding fragmentthereof, including combinations thereof, versus a different agent orantibody or antigen-binding fragment thereof. This type of prognosticand diagnostic monitoring and assessment is already in practiceutilizing antibodies against the HER2 protein in breast cancer(HercepTest™, Dako) where the assay is also used to evaluate patientsfor antibody therapy using Herceptin®. In vivo applications includedirected cell therapy and immune system modulation and radio imaging ofimmune responses.

In one embodiment, an anti-GITR antibody or antigen-binding fragmentthereof can be used in immunohistochemistry of biopsy samples.

In another embodiment, an anti-GITR antibody or antigen-binding fragmentthereof can be used to detect levels of GITR, or levels of cells whichcontain GITR on their membrane surface, which levels can then be linkedto certain disease symptoms. Anti-GITR antibodies or antigen-bindingfragments thereof described herein may carry a detectable or functionallabel. When fluorescence labels are used, currently available microscopyand fluorescence-activated cell sorter analysis (FACS) or combination ofboth methods procedures known in the art may be utilized to identify andto quantitate the specific binding members. Anti-GITR antibodies orantigen-binding fragments thereof described herein may carry afluorescence label. Exemplary fluorescence labels include, for example,reactive and conjugated probes e.g. Aminocoumarin, Fluorescein and Texasred, Alexa Fluor dyes, Cy dyes and DyLight dyes. An anti-GITR antibodyor antigen-binding fragment thereof may carry a radioactive label, suchas the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu,⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹¹⁷Lu, ¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁹⁸Au, ²¹¹At, ²¹³Bi,²²⁵Ac and ¹⁸⁶Re. When radioactive labels are used, currently availablecounting procedures known in the art may be utilized to identify andquantitate the specific binding of anti-GITR antibody or antigen-bindingfragment thereof to GITR (e.g., human GITR). In the instance where thelabel is an enzyme, detection may be accomplished by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques as knownin the art. This can be achieved by contacting a sample or a controlsample with an anti-GITR antibody or antigen-binding fragment thereofunder conditions that allow for the formation of a complex between theantibody or antigen-binding fragment thereof and GITR. Any complexesformed between the antibody or antigen-binding fragment thereof and GITRare detected and compared in the sample and the control. In light of thespecific binding of the antibodies described herein for GITR, theantibodies or antigen-binding fragments thereof can be used tospecifically detect GITR expression on the surface of cells. Theantibodies or antigen-binding fragments thereof described herein canalso be used to purify GITR via immunoaffinity purification.

Also included herein is an assay system which may be prepared in theform of a test kit for the quantitative analysis of the extent of thepresence of, for instance, GITR or GITR/GITRL complexes. The system ortest kit may comprise a labeled component, e.g., a labeled antibody, andone or more additional immunochemical reagents. See, e.g., Section 5.5below for more on kits.

5.5 Kits

Provided herein are kits comprising one or more antibodies describedherein, or antigen-binding fragments thereof, or conjugates thereof. Ina specific embodiment, provided herein is a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions described herein, such asone or more antibodies provided herein or an antigen-binding fragmentthereof. In some embodiments, the kits contain a pharmaceuticalcomposition described herein and any prophylactic or therapeutic agent,such as those described herein. In certain embodiments, the kits maycontain a T cell mitogen, such as, e.g., phytohaemagglutinin (PHA)and/or phorbol myristate acetate (PMA), or a TCR complex stimulatingantibody, such as an anti-CD3 antibody and anti-CD28 antibody.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

Also provided herein are kits that can be used in the above methods. Inone embodiment, a kit comprises an antibody described herein, preferablya purified antibody, in one or more containers. In a specificembodiment, kits described herein contain a substantially isolated GITRantigen (e.g., human GITR) as a control. In another specific embodiment,the kits described herein further comprise a control antibody which doesnot react with a GITR antigen. In another specific embodiment, kitsdescribed herein contain one or more elements for detecting the bindingof an antibody to a GITR antigen (e.g., the antibody can be conjugatedto a detectable substrate such as a fluorescent compound, an enzymaticsubstrate, a radioactive compound or a luminescent compound, or a secondantibody which recognizes the first antibody can be conjugated to adetectable substrate). In specific embodiments, a kit provided hereincan include a recombinantly produced or chemically synthesized GITRantigen. The GITR antigen provided in the kit can also be attached to asolid support. In a more specific embodiment, the detecting means of theabove described kit includes a solid support to which a GITR antigen isattached. Such a kit can also include a non-attached reporter-labeledanti-human antibody or anti-mouse/rat antibody. In this embodiment,binding of the antibody to the GITR antigen can be detected by bindingof the said reporter-labeled antibody.

The following examples are offered by way of illustration and not by wayof limitation.

6. EXAMPLES

The examples in this Section (i.e., Section 6) are offered by way ofillustration, and not by way of limitation.

6.1 Example 1: Generation of Novel Antibodies Against Human GITR

This example describes the generation and characterization of murineantibodies that bind to human GITR. In particular, this exampledescribes the generation of murine antibodies that specifically bind tohuman GITR and exhibit a co-stimulatory effect on CD4⁺ T cells.

To generate novel GITR antibodies, mouse CMS5a cells transfected withhuman GITR were used as immunogen in combination with an adjuvant(monophosphoryl lipid A (MPL), trehalose dimyocloate (TDM), muramyldipeptide (MDP) and freund's adjuvant (FA)) in BALB/c mice. Spleen cellsfrom immunized mice were fused with mouse myeloma cell line SP2/0.Supernatants of newly generated clones were screened in mixedhemadsorption assays on hGITR-transfected CMS5a and wild type CMS5acells. Selected supernatants were further tested by ELISA on recombinanthGITR protein (HGITR-Fc, Sigma). Fusion #231 resulted in four hybridomas(231-32-15, 231-1039-45, 231-1042-7 and 231-1333-21) with selectivereactivity for human GITR (sometimes referred to herein hGITR or huGITR)by MHA and ELISA. The antibodies (all IgG₁) were purified by protein Gaffinity chromatography for further testing.

The specificity of the anti-GITR-antibodies was tested by Western blotagainst purified recombinant human GITR, recombinant mouse GITR, CMS5acells transfected with human GITR, wild-type CMS5a cells, activated CD4⁺T cells and untreated CD4⁺ T cells. The Western blot, under non-reducingconditions, is depicted in FIG. 1. The anti-GITR antibodies werereactive with purified recombinant human GITR, not reactive withrecombinant mouse GITR, reactive with recombinant human GITR in CMS5acells, and reactive with natural human GITR in activated CD4⁺ T cells.

Analysis of ligand (GITR-L) and monoclonal antibody binding toimmobilized huGITR was performed by surface plasmon resonance andmeasured on BIAcore®. GITR (˜1100 RU) was immobilized onto a CM5 sensorchip using standard amine coupling. Analytes were injected over theimmobilized GITR for 15 minutes at a flow rate of 5 μl/min followed by a10 minute dissociation period. After kinetic runs had been performed theaffinity and dissociation constants were simultaneously calculated byBiaEvaluation® software (Biacore Life Sciences). Binding of human GITR-Lwas analysed over a concentration range of 12.5-200 nM and murineanti-GITR monoclonal antibodies were analysed over a concentration of6.25-100 nM. The antibodies did not bind to immobilized mouse GITR.Affinity and dissociation constants for the antibodies are tabulatedbelow in Table 9.

TABLE 9 Analyte K_(A) (1/M) K_(D) (M) huGITR-L 1.81 × 10⁸ 5.54 × 10⁻⁹mAb 231-1039-45 4.20 × 10⁸ 2.38 × 10⁻⁹ mAb 231-32-15 4.04 × 10⁸ 2.47 ×10⁻⁹ mAb 231-1333-21 4.19 × 10⁸ 2.39 × 10⁻⁹ mAb 231-1042-07 4.30 × 10⁸2.33 × 10⁻⁹

Hybridomas 231-32-15, 231-1039-45, 231-1042-7 and 231-1333-21 weresequenced and found to have the same cDNA and protein sequences. Theprotein sequences of the VH and VL were confirmed by N-terminal proteinsequencing and mass spectrometry (MS) of tryptic digests. The variableheavy chain sequence of the anti-GITR antibodies is SEQ ID NO: 201 andthe variable light chain sequence of the anti-GITR antibodies is SEQ IDNO: 202. The heavy chain variable region (VH) complementaritydetermining region (CDR) sequences VH CDR1, VH CDR2 and VH CDR3 have SEQID NOS: 13, 14 and 15, respectively and the light chain variable region(VL) CDR sequences VL CDR1, VL CDR2 and VL CDR3 have SEQ ID NOS: 16, 17and 18, respectively.

Competitive binding of the new 231-GITR antibodies was compared to acommercially available anti-GITR monoclonal antibody (R&D Systems mAb689clone 110416). GITR transfected CMS5a cells were used and incubated withan unlabeled anti-GITR mAb, followed by addition of PE-conjugated R&DmAb or Alexa 488-conjugated 231-1039-45 or 213-1333-21 antibody (datanot shown for Alexa 448-conjugated 213-1333-21 antibody), with resultsassessed by FACS analysis. Blocking studies of the R&D systems anti-GITRmAb are depicted in FIG. 2A and blocking studies of a 231 antibody(1039-45) are depicted in FIG. 2B. In these studies, either no antibody,unlabeled R&D antibody or unlabeled test antibodies (antibodies 1042-7,1039-45, 1331-21 or 32-15) were first added and incubated with M5Sa-GITRtransfected cells. This was then followed by addition of labeled R&Dsystems antibody (FIG. 2A) or the labeled 231-anti-GITR antibody 1039-45(FIG. 2B). The new 231 antibodies (1042-7, 1039-45, 1333-21 or 32-15)only partially blocked the binding of the R&D antibody, possibly due tosteric hindrance (FIG. 2A). FIG. 2B shows that the R&D antibody did notblock the binding of the 231-1039-45 antibody. Binding of 1039-45 wasinhibited by any of the 231 antibodies (i.e., by 1042-7, 1039-45,1333-21 or 32-15).

The binding characteristics of the 231-GITR antibodies were analyzed byFACS and the results are depicted in FIGS. 3A-C. FIG. 3A shows stainingof CMS5a-GITR cells with anti-human GITR IgG₁ 1333-21 antibody from twodifferent batches (1 and 2) and R&D Systems antibody. In FIG. 3B,fluorescence intensity of ex-vivo PMBC derived CD3⁻CD19⁻ GITR⁺ andCD4⁺CD25⁺ GITR⁺ is shown upon staining with 1042-7, 32-15, 1039-45,1333-21 and R&D Systems antibodies. FACS analysis of these in vivo PBMCderived cells after 1333-21 or R&D systems antibody (mAb689 clone110416) binding is shown in FIG. 3C.

Next, studies were performed to assess the costimulatory effect of theanti-GITR antibody on CD4⁺ T cells in combination with anti-CD3 (OKT3)antibody. The most significant relative costimulatory effect wasobserved by combining anti-GITR antibodies (231-1042-7, 231-32-15,231-1039-45, 231-1333-21) with a suboptimal concentration (0.2 μg/ml) ofOKT3. Anti-GITR antibody (5 μg/ml) and OKT-3 antibody at varyingconcentrations were bound to tissue culture plates and then incubatedwith CSFE-labeled CD4⁺ cells. IL-2 (10 U/ml) was added to the medium andthe cells and antibodies were incubated a further 5 days. At the end of5 days, CFSE intensity was evaluated with divided cells having low CFSEintensity. IFNγ was also measured. The results for the anti-GITRantibodies are depicted in FIG. 4. At a suboptimal concentration of OKT3antibody (0.2 μg/ml), the anti-GITR antibodies had a significantrelative effect on proliferation of CD4⁺ cells. An increase in IFNγcompared to controls was seen in the presence of the anti-GITR antibodycombined with all levels of OKT3 tested, with the most pronounced effectobserved at 0.2 μg/ml and 0.04 μg/ml. Variation between the antibodiestested can be attributable to intra-assay variability and a naturalvariability between each antibody preparation (e.g. separatepreparation). Notably, in the absence of OKT3, there is no stimulationby the anti-GITR antibodies. The costimulatory effect of the anti-GITRantibodies with OKT3 was dose dependent on the amount of the anti-GITRantibodies (data not shown).

In conclusion, new anti-GITR antibodies have been isolated that arespecific for human GITR and recognize recombinant and natural GITR.These antibodies bind hGITR expressed on hGITR-transfected CMS5a cellsat 2.5-5 ng/ml and show good binding by FACS analysis. The antibodiesalso bound to human PBMC including T cells expressing natural hGITR.Their affinity for GITR by BIAcore® is K_(A) (1/m) approximately4.2×10⁸, K_(D) (m) 2.4×10⁻⁹ (versus GITRL (ligand) K_(A) of 1.8×10⁸ andK_(D) 5.5×10⁻⁹). The antibodies bind to a different site on hGITR versusa commercially available GITR mAb (R&D).

A costimulatory effect of anti-GITR monoclonal antibody with suboptimalconcentrations (0.2 μg/ml and 0.04 μg/ml) of anti-CD3 mAb (OKT3) on CD4⁺T cells has been demonstrated. Despite the presence of T-regulatorycells in the population of enriched CD4⁺ T cells, the anti-GITRantibodies enhanced CD4 T cell activity.

6.2 Example 2: Humanization of Mouse Monoclonal Antibody 231-32-15

This example describes the humanization of the murine antibody 231-32-15and the characterization of the humanized antibodies.

6.2.1 Humanization of Murine Antibody 231-32-15

Humanization of the anti-human GITR mouse antibody 231-32-15 includingselection of human acceptor frameworks is described herein.

6.2.2 Chimerization of Murine Antibody 231-32-15

The murine VH and VL (kappa) variable regions from murine 231-32-15having SEQ ID NOS: 201 and 202, respectively, were synthesized byGeneArt® (Life Technologies™). The natural leader sequences from theoriginal murine variable domains were included along with adapters withrestriction sites to allow the cloning of these variable regionsdirectly into standard in-house human IgG₁ Vh vector (CH1-2-3 domains),and Vk vector (Ck1), to create chimeric genes with a murine Vh or Vk,and human constant regions. The chimeric heavy and light chainexpression vectors were then co-transfected into CHO cells in suspensionto produce chimeric antibody protein for use in the assays performedbelow. This chimeric antibody is referred to in the Examples in Section6 as the “chimeric parental 231-32-15 antibody”. This chimeric parental231-32-15 antibody contains a T109S substitution (i.e., substitution ofthreonine with serine at position 109 relative to the wild type Fcsequence), numbered according to Kabat, in the light chain constantdomain, which facilitates the cloning of the variable region in frame tothe constant region. This mutation is a conservative modification thatdoes not affect antibody binding or function.

Homology matching was used to choose human acceptor frameworks to graftthe CDRs of antibody 231-32-15. Databases, e.g., a database of germlinevariable genes from the immunoglobulin loci of human and mouse (the IMGTdatabase (the international ImMunoGeneTics information System®; LefrancM P et al., (1999) Nucleic Acids Res 27(1): 209-12; Ruiz M et al.,(2000) Nucleic Acids Res 28(1): 219-21; Lefranc M P (2001) Nucleic AcidsRes 29(1): 207-9; Lefranc M P (2003) Nucleic Acids Res 31(1): 307-10;Lefranc M P et al., (2005) Dev Compo Immunol 29(3): 185-203; Kaas Q etal., (2007) Briefings in Functional Genomics & Proteomics 6(4): 253-64)or the VBASE2 (Retter I et al., (2005) Nucleic Acids Res 33, Databaseissue D671-D674) or the Kabat database (Johnson G et al., (2000) NucleicAcids Res 28: 214-218)) or publications (e.g., Kabat E A et al., supra)may be used to identify the human subfamilies to which the murine heavyand light chain variable regions belong and determine the best-fit humangermline framework to use as the acceptor molecule. Selection of heavyand light chain variable region sequences (VH and VL) within thesesubfamilies to be used as acceptor may be based upon sequence homologyand/or a match of structure of the CDR1 and CDR2 regions to helppreserve the appropriate relative presentation of the six CDRs aftergrafting.

Searching of the IMGT database using IgBLAST (available on the NCBIwebsite) indicated good homology between the 231-32-15 heavy chainvariable region framework and members of the human heavy chain variableregion subfamilies 1 and 7. Highest homologies and identities of bothCDR and framework sequences were observed for germline sequence:IGHV1-2*02 (also known as DP75; SEQ ID NO: 601) (59.2% identity; 58amino acid residues out of 98), IGHV1-3*01 (SEQ ID NO: 602) (58.2%identity; 57/98), IGHV1-46*01 (SEQ ID NO: 603) (57.1% identity; 56/98),IGHV1-18*01 (SEQ ID NO: 604) and IGHV1-69*01 (SEQ ID NO: 605) (both56.1% identity; 55/98) and IGHV7-4-1*02 (SEQ ID NO: 606) (54.1%identity; 53/98).

Using the same approach, 231-32-15 light chain variable domain sequenceshowed good homology to the members of the human light chain variabledomain kappa subfamilies 3 and 4. Highest homologies and identities ofboth CDRs and framework sequences were observed for germline sequences:IGKV4-1*01 (SEQ ID NO: 607) (79.2% identity; 80 amino acid residues outof 101) and IGKV3-7*02 (SEQ ID NO: 608) (64.4% identity; 65/101).

As a starting point to the humanization process, a CDR grafted versionof mouse 231-32-15 VH was generated using human IGHV1-2*02 as humanframework acceptor. A number of back mutations were made at residuepositions that may affect the conformations of CDRs or inter-variabledomain packing and therefore may be structurally important formaintaining the full activity of the antibody. In framework 1, residueKabat H24 was kept as mouse because it is a canonical residue for CDR1(loop of 5 amino acid residue long as defined by Kabat). It is a Gly inthe murine sequence and an Ala in the human germline. In framework 2,residue Kabat H48 was kept mouse because it is known as a Vernierresidue (i.e., in close proximity to CDRs). It is an Ile in the murinesequence and a Met in the human germline. In framework 3, residues KabatH67 and 73, which are Vernier residues were kept mouse. H67 is an Ala inthe murine sequence and Val in human IGHV1-2*02 germline. H73 is Lys inthe murine sequence and Thr in human IGHV1-2*02 germline. Residue H71,which is a critical canonical residue for CDR2 has been kept mouse (itis an Arg in human germline and a Val in the murine sequence). ResidueKabat H94, which is a canonical residue for CDR1 has been kept mouse (itis an Arg in human germline and a Lys in the mouse sequence). The finalhumanized sequence was termed Version VH A (SEQ ID NO: 206) and had79.6% identity (78 amino acid residues out of 98) with IGHV1-2*02 humangermline.

A first CDR grafted version of mouse 231-32-15 VL was generated usinghuman IGKV3-7*02 as human framework acceptor. Back mutations wereconsidered at various residue positions and as a result in framework 3,residue Kabat L87 was kept mouse since it might play a critical role inthe VH/VL interface. L87 is a His in the murine sequence and Tyr inIGKV3-7*02 human germline. The final humanized sequence was termedVersion VK A1 (SEQ ID NO: 207) and had 81.2% identity (82 amino acidresidues out of 101) with IGKV3-7*02 human germline.

A second CDR grafted version of mouse 231-32-15 VL was generated usinghuman IGKV4-1*01 as human framework acceptor. Back mutations wereconsidered at various residue positions and as a result in framework 3,residue Kabat L87 was kept as mouse since it might play a critical rolein the VH/VL interface. L87 is a His in the murine sequence and Tyr inIGKV4-1*01 human germline. The final humanized sequence was termedVersion VK A2 (SEQ ID NO: 208) and had 91.1% identity (92 amino acidresidues out of 101) with IGKV4-1*01 human germline.

Table 10 shows residues (Kabat numbering) that differ between mouse andhuman antibody frameworks in the CDR grafted versions of mouse 231-32-15VH and VL described above.

TABLE 10 Comparison of 231-32-15 and the human acceptor heavy chainvariable IGHV1-2*02 and human acceptor light chain variable IGKV4-1*01and IGKV3-7*02 framework. Heavy Chain Variable Region Kabat position231-32-15 IGHV1-2*02 24 Gly Ala 48 Ile Met 67 Ala Val 71 Val Arg 73 LysThr 94 Lys Arg Light Chain Variable Region IGKV4-1*01/ Kabat position231-32-15 IGKV3-7*02 87 His Tyr

6.2.3 Expression of Humanized Variants

The variable regions of IGHV1-2*02, IGK4-1*01 and IGK3-7*02 weresynthesized by Life Technologies™ and cloned in standard expressionvectors (pPEP), as described below. These constructs were subsequentlyused to transfect CHO cells and the expressed antibodies were testedusing suspension array technology (Luminex® 200 system, Millipore) andsurface plasmon resonance (BIAcore®, GE Healthcare) as described below.The term “variable region” in this Example means VDJ rearranged genesfor the heavy chains IGHV1-2*02 and VJ rearranged genes for the lightchains IGK4-1*01 and IGK3-7*02. The expression vector contained a CMVpromoter, immunoglobulin constant region, WPRE element(posttranscriptional response element) and a BGH polyadenylation signal.Three different expression vector variants were used for cloning of thevariable region IGHV1-2*02. Two expression vector variants containeddifferent immunoglobulin constant regions—IGHG1 and IGHG4 and the thirdexpression vector variant contained a fragment of IGHG1 to produceantibody Fab fragments. The variable regions of light chains IGK4-1*01and IGK3-7*02 were cloned in an expression vector containing the IGKCimmunoglobulin constant region.

6.2.3.1 Cloning in Expression Vectors for CHO Cell Transfection

Synthesised variable regions were cloned into a pPEP expression vectorcontaining the appropriate immunoglobulin constant region. For thecloning of heavy chain variable regions of IGHV1-2*02, constructs 3592(pPEP-InsX-Cg(iso3), 4192 (pPEP-InsX-IgG₄) and 4215(pPEP-InsX-Fab-Xa-6×His) (“6×His” disclosed as SEQ ID NO: 36) weredigested with HindIII/Eco47III at 37° C. for 4 hours. After digestion,the bands with a size of 4952 bp, 4952 bp and 4313 bp were gel-purified(Macherey & Nagel, NucleoSpin Gel and PCR cleanup). For the cloning ofkappa light chain variable regions IGK4-1*01 and IGK3-7*02, construct3593 (pPEP-Ins-Ck) was digested with HindIII/Eco47III at 37° C. for 4hours and the band with a size of 4474 bp was gel-purified. Thesynthesized antibody variable regions were digested withHindIII/Eco47III at 37° C. for 4 hours and the bands with a size of 422bp (IGHV1-2*02) and 411 bp (IGK4-1*01 and IGK3-7*02) were gel-purified.

The digested and purified variable antibody regions (IGHV1-2*02,IGK4-1*01, IGK3-7*02) were ligated in-frame into a pPEP vectors (50 ng)containing the appropriate immunoglobulin constant regions. Ligation wasperformed overnight at 16° C. with a vector to insert ratio of 1:3.Afterwards 1 μl of ligation reaction was electroporated into DH10B cells(E. coli ElectroMax DH10B electrocompetent cells, Invitrogen) (1900V/5ms). Then 5-50 μl of the electroporated bacteria were plated ontoLB-agar+100 μg/ml ampicillin plates. From each construct about 2-3colonies were picked and grown overnight.

From each clone a DNA plasmid preparation in a small-scale was performed(Macherey & Nagel, NucleoSpin Plasmid). A digestion to verify thepresence and the correct size of the cloned insert was performed withHindIII/Eco47III (H/E) and an ApaLI (A) digestion was used to verify thecorrect vector backbone. The vector integrity was tested by separationof uncut plasmid DNA. From each positive clone and DNA, the preparationwas up-scaled, control digested and sequenced using primer 892-Je(Sequence: 5′ gaccaatagaaactgggcttgtc 3′; SEQ ID NO: 705). Eachconstruct received a construct number, as a unique identifier andglycerol stock was prepared.

TABLE 11 Construct characterization in expression vectors Constructcharacterization Construct number Insert Immunoglobulin constant regions4260 IGHV1-2*02 IGHG1 4261 IGK3-7*02 IGKC 4262 IGK4-1*01 IGKC 4379IGHV1-2*02 IGHG4 4336 IGHV1-2*02 IGHG1-Fab

6.2.3.2 Cloning in Retroviral Expression Vectors

The variable regions of IGHV1-2*02, IGK3-7*02 and IGK4-1*01 weresynthesised by Life Technologeis™ and cloned into retroviral expressionvectors (pCMA). These constructs were subsequently used to transducepreB cells and to express antibodies on the surface using RetrocyteDisplay® technology. The retroviral expression vector contained MSCVbased 5′ and 3′LTR's, immunoglobulin constant region (IGHG1 or IGKC)comprising membrane anchor fraction (IGHG1) and a CD8 surface markergene.

Synthesised variable regions were cloned into a retroviral expressionvector containing the appropriate immunoglobulin constant region. Forthe cloning of the heavy chain variable region, construct 3956(pCMA-InsX Cg(iso3) loxP2-I-tr_huCD8-loxP) was digested withHindIII/Eco47III at 37° C. for 4 hours and the band of size of 7616 bpwas gel-purified. For the cloning of kappa light chain variable regions,construct 3957 (pCMA-InsX Ck-I-tr_huCD8) was digested withHindIII/Eco47III at 37° C. for 4 hours and the band of size of 6718 bpwas gel-purified. The synthesized antibody variable regions weredigested as described in Section 6.2.1 above.

The digested and purified variable antibody regions were ligatedin-frame into retroviral expression vectors (50 ng) containing theappropriate immunoglobulin constant regions. Ligation, transformationand clone verification were performed as described in Section 6.2.3.1above. The up-scaled DNA plasmid preparations were sequenced usingprimer 327-Je (Sequence: 5′ ctcgatcctccctttatccag 3′; SEQ ID NO: 706).Each construct received a construct number, as a unique identifier andglycerol stock was prepared.

TABLE 12 Construct characterization in retroviral expression vectorsConstruct characterization Construct Immunoglobulin number Insertconstant regions Surface marker 4257 IGHV1-2*02 IGHG1 CD8 4258 IGK3-7*02IGKC CD8 4259 IGK4-1*01 IGKC CD8

6.2.4 Expression of Recombinant Antibodies

Recombinant antibodies were expressed by transient transfection ofFreeStyleCHO-S cells (Invitrogen, R800-07) in suspension. Briefly, celldensity was adjusted to 8×10⁶ cell/ml in PowerCHO2 medium (Lonza,12-771Q) supplemented with 4 mM L-glutamine (Biochrom, K 0283) and 1×HTsupplement (GIBCO, 11067-030). DNA corresponding to antibody light chain(2.5 μg/ml) and heavy chain (2.5 μg/ml) was added to the cell suspensionwith slight agitation. Following DNA addition, the cell suspension wassupplemented with 10 μl/ml of TransIT-Pro transfection reagent (MIRUS,MIR5700) and 0.5 mM (final concentration) valproic acid (Sigma-Aldrich,P4543). The cell suspension was incubated for 6 days at 31° C., 8% CO₂with shaking (200 rpm).

The culture supernatant was harvested by centrifugation (9000 g, 10 minat 10° C.) and filtered through a 0.45 μm filter. A Vivaspin 20ultrafiltration device (Sartorius, VS2032, MWCO 50 kDa) was used toconcentrate the supernatant to final volume of 0.6 ml at 1000 g, 10° C.for approximately 60 minutes. For purification of recombinant antibody aprotein A HP Spin Trap column (GE Healthcare, 28-9031-32) wasequilibrated with binding buffer (20 mM phosphate buffer pH 7.0) and 0.6ml of concentrate was loaded. The column was sealed with a lid andincubated on an end-over-end mixer at room temperature. After 30 min,the column was washed 2× by application of 600 μl binding buffer andsubsequent centrifugation at 100 g for 1 minute. Bound recombinantantibody was eluted from the spin column by adding 400 μl elution buffer(100 mM glycine, pH 2.0) and centrifugation at 100 g for 1 minute.Eluates were immediately neutralised with 40 μl of neutralisation buffer(1M Tris-HCl, pH 9.0). Purified recombinant antibody was stored inprotein LoBind tubes (Eppendorf, 0030 108.116) at 4° C. until furtherprocessing for characterisation studies.

For quantification, cell culture supernatants and purified samplescontaining human IgG were diluted in assay buffer (Roche, 11112589001)and dilutions were assessed in duplicate in a 96-half well plate(Corning, 3884). Briefly, 25 μl samples were incubated in the dark (20°C., 650 rpm) for 1 hour with 5 μl containing 1200 Luminex-COOH-beadsloaded by amine coupling with goat anti-human IgG, F(ab′)₂ fragmentspecific (Jackson ImmunoResearch, 109-006-09). Standard curves weregenerated using duplicates of 25 μl of a 1:3 dilution series (0.08-60ng/ml) of ChromPure human IgG whole molecule (Jackson ImmunoResearch,009-000-003). Detection was performed by the addition of 30 μlanti-human IgG, Fc-specific labeled with R-PE (5 μg/ml; JacksonImmunoResearch, 109-116-098) and further incubation for 1 h. Plates wereread and analysed using a Luminex® 200 instrument (Millipore) using thefollowing settings: 100 beads, 50 μl sample size.

Quality control of purified samples included analysis by SDS-PAGE andsize exclusion chromatography (SEC) on HPLC. SDS-PAGE analysis wasperformed under reducing and non-reducing conditions on 12% and 8%polyacrylamide gels, respectively, following the protocol of Laemmli(Laemmli U K (1970) Nature, 227(5259): 680-5). Polyacrylamide gels werestained with Commassie brilliant blue solution for visualization. SECwas performed on an Agilent Infinity 1260 system (Agilent Technologies)equipped with a binary pump, degassing unit, automatic sampling unit andUV diode array detector. For separation Zenix-C 300 column (3 μmparticle size, 4.5×300 mm, Sepax, 233300-4630) was used. 3 μg of eachsample was loaded and separated in 100 mM phosphate buffer pH 7.0(supplemented with 150 mM NaCl) at 0.35 ml/min for 15 minutes anddetection was at 220 nm.

6.2.5 Characterization of Humanized Variants

The binding properties of both humanized variants (Hum231#1: IGHV1-2*02and IGK3-7*02; Hum231#2:IGHV1-2*02 and IGK4-1*01) and the chimericparental antibody 231-32-15 were characterized in a number of assays asdescribed below.

6.2.5.1 Quantification and Binding Analysis by Suspension ArrayTechnology

Purified material of both humanized variants and the chimeric parentalantibody 231-32-15 was diluted in assay buffer (Roche 11112589001)1:10,000 and 1:100,000. Briefly, 25 μl of each dilution was incubated inthe dark (20° C., 650 rpm) with 1500 Luminex® beads (in 5 μl assaybuffer) for 1 hour in 96 half-well filter plates (Millipore, MABVN1250).Luminex® beads (Luminex Corp, #5 LC10005-01 and #14 LC10014-01) wereeither coupled with anti-human IgG (F(ab)₂-specific, JIR, 105-006-097)or GITR antigen (R&D systems, di-sulfide-linked homodimer; 689-GR) viaamine coupling with COOH bead surface. Standard curves were generatedusing duplicates of 25 μl of ChromPure IgG whole molecule (JIR,009-000-003) with 1:3 dilution series (0.08-540 ng/ml) for IgG₁versions. For antibodies in IgG₄ format, a different standard wasused—purified Immunoglobulin (Sigma, 14639). Detection was performedusing 60 μl of goat anti-human IgG F(ab)₂ labeled with R-PE (2.5 μg/ml;JIR 109-116-098, AbDSerotec Rapid RPE Antibody Conjugation Kit,LNK022RPE) and 1 hour of incubation time (20° C., 650 rpm). Plates wereanalyzed using a Luminex® 200 system (Millipore). A number of 100 beadswere counted per well in 48 μl sample volume. Relative affinities werecalculated in relation to the MFI values of the chimeric parental231-32-15 antibody (set to 100% binding) and according to IgG valuespresent in the sample. Both humanized variants showed relativeaffinities near to 100%.

6.2.5.2 Ligand Blocking Activity Using Suspension Array Technology

To determine whether anti-GITR antibodies blocked binding of ligand(GITRL) to GITR, a ranking assay setup was performed using suspensionarray technology. 1200 Luminex® beads in 5 μl assay buffer (LuminexCorp, #14 LC10014-01) were added to each well of 96-well half areaplates (Corning, Inc., 3884). The beads were coupled with GITR antigen(R&D systems, di-sulfide-linked homodimer; 689-GR) via amine couplingwith COOH bead surface. The coupling reaction was performed using 50μg/ml of GITR antigen and 1×10⁷ Luminex beads per ml. Standard NHS esterchemistry was used to form carbodiimide bonds between the primary aminegroups of the antigen and the carboxyl groups on the bead surface(Luminex Xmap cookbook chapter 3).

Antigen coupling for proteins is a simple two-step carbodiimideprocedure during which microsphere carboxyl groups are first activatedwith EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)reagent in the presence of Sulfo-NHS (N-hydroxysulfosuccinimide) to forma sulfo-NHS-ester intermediate. The reactive intermediate is thenreplaced by reaction with the primary amine of the target molecule(antibody, protein or peptide) to form a covalent amide bond. Thecoupled beads were incubated with different concentrations of anti-GITRantibodies (concentrations of 9000 ng/ml to 12 ng/ml in 25 μl assaybuffer per well) for 1 h at 20° C. and 650 rpm. Afterwards 30 μl of R-PElabeled GITR-ligand (concentration 1 nM; monomeric, R&D systems694-GF/CF) was added to each well, giving a total well volume of 60 μl(1200 beads per well and a final concentration of 0.5 nM of labeledGITRL). The labeling of the ligand was done in-house using R-PE labelingkits (AbDSerotec, LYNX Rapid RPE Antibody Conjugation Kit, LNK023RPE)according to the manufacturer's protocol. Plates were analyzed using aLuminex® 200 system (Millipore). A number of 100 beads were counted perwell in 50 μl sample volume. Ligand blocking potential was calculatedusing the MFI values of the non-competed signal (100% binding) of theligand only control. A PE detectable signal indicated ligand binding tothe antigen.

In a first assay, the anti-GITR antibodies chimeric parental 231-32-15and m6C8 (WO 06/105021) as well as a control antibody recognising IL-1β(SK48E26; International Publication No. WO 95/001997) were tested. Theantibody m6C8 was an IgG1 antibody generated based on the variableregions of the antibody 6C8 provided in WO 06/105021 (hereinincorporated by reference). The heavy chain of m6C8 comprises the aminoacid sequence of SEQ ID NO: 585. The light chain of m6C8 comprises theamino acid sequence of SEQ ID NO: 586. Results of this assay are shownin FIG. 5, where it can be observed that at concentrations of the 6C8antibody above 333 ng/ml, no PE signal was detected and therefore nobinding of GITRL to GITR occurred. In contrast, for the chimericparental 231-32-15 antibody, at all concentrations tested, a PE signalwas detected indicating that GITRL was still able to bind to GITR whenthe chimeric parental 231-32-15 antibody was also bound to GITR. Thedata shown in FIG. 5 are from four repeats of this assay, performed induplicate, with the standard deviation calculated for n=8.

In a second assay, GITRL-PE binding to GITR was tested in the presenceof the chimeric parental 231-32-15 anti-GITR antibody and the humanizedvariants Hum231#1 and Hum231#2. FIG. 6 shows that these three anti-GITRantibodies when bound to GITR, still allow the binding of GITRL to GITRand all three antibodies show comparable ligand blocking activity.

6.2.5.3 Kinetic Analysis by Surface Plasmon Resonance

Surface plasmon resonance was used to determine the affinity of thehumanized variants and the chimeric parental 231-32-15 antibody(BIAcore® T100/T200 sensitivity enhanced system (GE Healthcare) and aFab-capture assay). All interactions were analyzed at 25° C. using1×DPBS (PAA, H15-002) plus P20 (0.05%, Pierce, 28320) as running buffer.Anti-GITR antibodies (8 μg/ml in running buffer) were captured to thechip surface of a CM5 sensor chip (GE Healthcare, Series S CM5,BR-1005-30) via an immobilized anti-human Fab antibody (GE Healthcare,Fab Capture Kit, 28958325). To detect unspecific interactions of theGITR antigen, antibody capture was only performed in flow cell 2,whereas in flow cell 1 only the capturing antibody was immobilized. Inaddition, an unrelated antibody (anti-IL-13; SK48E26; InternationalPublication No. WO 95/001997) was used to assess specificity of GITRbinding. After capture of the anti-GITR antibodies GITR antigen (R&Dsystems, di-sulfide-linked homodimer; 689-GR) was run through both flowcells in different amounts (40 nM, 10 nM and 2.5 nM) for each antibody.Also a blank curve (running buffer only) was included in each run.Association was run for 90 s and dissociation for 600 s with a flow rateof 10 μl/min. After each run a regeneration step was performed with 10mM Glycine pH2.0 (GE Healthcare, BR-1003-55) for 60 s with 30 μl/min.Binding curves were evaluated using BIAcore® T200 evaluation softwareversion 2.0.1 applying a Langmuir 1:1 model with global fit of Rmax.

From these values an affinity value (K_(D) (M)) was calculated and thevalues are shown in Table 13 below. The humanized variants Hum231#1 andHum123#2 showed improved on-rates but decreased off-rates resulting inK_(D) values of 0.7 nM and 0.6 nM, respectively. The chimeric parental231-32-15 antibody had an affinity of 2 nM.

TABLE 13 Summary of on- and off-rates and calculated K_(D) (M) anti-GITRantibody k_(on) (1/Ms) k_(off) (1/s) K_(D) (M) Chimeric parental231-32-15 3.52E+05 7.12E−04 2.02E−09 Hum231#1 3.55E+06 2.49E−03 7.02E−10Hum231#2 2.83E+06 1.78E−03 6.29E−10

6.2.5.4 Ligand Blocking Analysis by Surface Plasmon Resonance

Both humanized variants Hum231#1 and Hum231#2 were expected to show thesame ligand blocking kinetics as the chimeric parental 231-32-15antibody. This was confirmed using a ligand blocking assay as measuredby surface plasmon resonance (BIAcore® T100/T200 sensitivity enhancedsystem (GE Healthcare)).

In a first experiment binding of GITR ligand to immobilized GITR antigenwas evaluated. GITR antigen (R&D systems, disulfide-linked homodimer;689-GR) was immobilized at high density (4371 RU) on a CM5 sensor chip(GE Healthcare, Series S CM5, BR-1005-30). In another flow cellovalbumin (1289 RU, Pierce ThermoFisher 77120) was immobilized forreference. Immobilization was performed according to a standard protocolfrom the manufacturer (GE Healthcare) for amine coupling (activation ofsurface with 0.4 M EDC and 0.1 M NHS, GE Healthcare Amine coupling kit,BR-1000-50). Unreacted groups were inactivated with 1M ethanol-amine-HClpH 8.5. Afterwards two GITR ligands (monomer R&D, 694-GL andnon-covalently linked homotrimer R&D, 6987) were run through the chipsurface in different amounts (500 nM, 250 nM and 125 nM) to determinesaturating conditions. An association time of 240 s and a dissociationtime of 300 s were used with a flow rate of 5 μl/min. Regeneration ofthe chip surface was done using 10 mM Glycine pH2.0 (GE Healthcare,BR-1003-55) for 60 s at 10 μl/min. Most favourable saturating conditionswere achieved with the GITR trimeric ligand at 125 nM and this setup wastherefore used with anti-GITR antibodies at the same amount. In anotherexperiment, the reverse setup was used so that the anti-GITR antibodies(125 nM) were first bound to the GITR antigen on the chip and the GITRligand (non-covalently linked trimer at 125 nM) was added afterwards.

As is shown in FIG. 7, when GITR antigen was immobilized on the chip andGITRL was added in the presence of the anti-GITR antibodies chimeric231-32-15 antibody, Hum231#1 and Hum231#2, binding of GITRL wasobserved. In contrast, no binding of GITRL was observed in the presenceof the anti-GITR antibody m6C8. These data indicate that chimeric231-32-15 antibody, Hum231#1 and Hum231#2 do not inhibit human GITR frombinding to GITRL.

6.3 Example 3: Functional Characterisation of Humanized Antibodies

This example demonstrates the ability of the humanized anti-GITRantibodies generated by the methods described above to function as anagonist of GITR. The anti-GITR antibody Hum231#2 comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 567 and a light chaincomprising the amino acid sequence of SEQ ID NO: 587. The antibodyHum231#2 is a human IgG1 antibody containing a T109S substitution (i.e.,substitution of threonine with serine at position 109 relative to thewild type Fc sequence), numbered according to Kabat, in the light chainconstant domain, which facilitates the cloning of the variable region inframe to the constant region. This mutation is a conservativemodification that does not affect antibody binding or function. The wildtype counterpart, named Hum231#2w, which contains a threonine atposition 109, numbered according to Kabat, was also generated. Theantibody Hum231#2w is a human IgG1 antibody comprising a heavy chain ofSEQ ID NO: 567 and a light chain of SEQ ID NO: 576.

These anti-GITR antibodies were also assayed to determine their abilityto costimulate primary human CD4⁺ or CD8⁺ T-cells. This work, asdescribed in Section 6.3.1 to 6.3.3 and 6.3.7 below, was performed withmaterials from multiple donors. Human leukocytes used in screening andtesting of candidate antibodies were procured from the New York BloodCenter (New York City).

The functional activity of anti-GITR antibodies was demonstrated onenriched CD4 positive (a.k.a. CD4⁺) T cells, CD8 positive (a.k.a. CD8⁺)T cells and PBMCs. For human T-cell proliferation studies, freshlyprepared, donor packed leukocytes were collected and processed usingsterile tissue culture techniques. Leukocytes were processed to harvestmononuclear immune cells (PBMC) by density gradient (LymphocyteSeparation Media, Corning). PBMCs are located in the buffy coat layer ofFicol density gradient.

Enriched CD4 cells were prepared from PBMC by negative selection usingRosetteSep® Human CD4⁺ T Cell Enrichment Cocktail (StemcellTechnologies, Vancouver, BC Canada). The enriched CD4⁺ T-cellpreparations were separated from red blood cells by densitycentrifugation over Lymphocyte separation media (Corning). Collectedcells were washed and aliquoted for storage in liquid nitrogen. Toaccount for variability in donor's response to stimulation, varyingconcentrations of anti-CD3 were used to adjust donor-specific capabilityto response. Therefore, prior to screening anti-GITR antibodies, thebuffy coats were assessed for their capability for cytokine release andproliferation in response to CD3 stimulation with titrated levels ofanti-CD3 (Clone SP34; BD Pharmingen; concentration ranging from 31.5ng/ml to 250 ng/ml), with or without reference anti-GITR chimericparental 231-32-15 antibody to establish baseline T-cell proliferationand their cytokine production and determine appropriate stimulationconditions for each donor buffy coat.

6.3.1 Effect of Agonistic Anti-GITR Antibodies on Anti-CD3 StimulatedCD4+ T Cell Proliferation

Anti-GITR antibodies were assessed for agonist activity by theircostimulation of CD4+ T cells. The agonistic activity of chimericparental 231-32-15 antibody was compared to the two humanized versions:Hum231#1 and Hum231#2. The costimulation assay was performed as follows:For plate bound stimulation conditions, anti-CD3 antibodies, anti-GITRantibodies and where indicated isotype control, were coated on flatbottom or round bottom sterile tissue culture plates for two hours andexcess antibodies were removed through washing. For solublecostimulation conditions, anti-CD3 antibody was coated onto a platewhile costimulation with anti-GITR antibodies was provided in solution.Tested anti-GITR antibodies were Hum231#1 and Hum231#2, chimericparental 231-32-15 antibody (a.k.a REF-231) or negative isotype controls(pAB1915). Additionally, for plate bound and soluble costimulationconditions, anti-CD28 antibody (125 ng/ml; BD Pharmingen) and 10 U L-2were also provided in solution.

Cellular proliferation was determined by monitoring dilution ofcarboxyfluorescein diacetate sucinimidyl ester (CFSE) dye within dividedcells (Quah B J et al., (2007) Nat Protoc, 2(9): 2049-56). The enrichedCD4⁺ T cells were labeled with 1-2 μM CFSE. CFSE-labeled CD4⁺ enriched Tcells were washed and then stimulated with plate bound anti-CD3 (125ng/ml), soluble anti-CD28 (125 ng/ml) and 10 U IL-2, together with 5μg/ml or 10 μg/ml plate bound anti-GITR antibodies or no antibody. Thecells were allowed to divide for 3 to 6 days in culture at 37° C.depending on optimal activation of each donor cell at which pointculture supernatants and cells were collected from the plate.

FIGS. 8A and 8B show a representative FACS analysis of CD4⁺ T cellproliferation induced by costimulation with anti-GITR antibodiesperformed in triplicate on buffy coat 6 and buffy coat 8, respectively.These figures show cell numbers (Y-axis) and the level of fluorescenceemitted (X-axis) by the CFSE labeled CD4⁺ T cells. 10 μg/ml anti-GITRantibodies (chimeric parental 231-32-15 (REF 231-32-15), Hum231#1 andHum231#2) were used. Enhanced CD4⁺ T cell proliferation is shown by anincreased percentage of cells with a diminished level of fluorescenceemitted by CFSE (CFSE low). FIGS. 8A and 8B illustrate that anti-GITRantibodies (chimeric parental 231-32-15 (REF 231-32-15), Hum231#1 andHum231#2) demonstrated agonistic activity when added to cells activatedwith suboptimal concentrations of anti-CD3 antibody for both highresponding cells (buffy coat 6, FIG. 8A) and even for low respondingcells (buffy coat 8; FIG. 8B).

FIGS. 9A and 9B are histogram plots of the representative results fromthe above study for plate bound anti-GITR antibodies (chimeric parental231-32-15 antibody, Hum231#1, Hum231#2, and m6C8) at a concentration of10 μg/ml. In the example shown in FIG. 9A, costimulation with Hum231#1or Hum231#2 induced CD4+ T cell proliferation at 10 μg/ml. Approximatelygreater than 50% of CD4⁺ T cells proliferated (CFSE low cells) whencostimulated with 10 μg/ml Hum231#1 or Hum231#2. In contrast, underanti-CD3/anti-CD28 stimulation without anti-GITR antibody-mediatedcostimulation, only approximately 35% of CD4⁺ T cells proliferated (CFSElow cells). FIG. 9B illustrates that the addition of anti-GITRcostimulation to anti-CD3/anti-CD28-mediated stimulation also caused anincrease in the absolute number of CD4⁺ T cells in culture over 5 daysas compared to stimulation with anti-CD3/anti-CD28 alone. For example,stimulation with anti-CD3/anti-CD28 in conjunction with 10 μg/mlantibody chimeric parental 231-32-15 antibody (REF 231) caused GITRcostimulation induced expansion of CD4⁺ T cell numbers from 7.5×10⁴ to12.0×10⁴.

Additionally, Hum231#1-mediated costimulation at 10 μg/ml also inducedexpansion of CD4⁺ T cells from 7.5×10⁴ to 11.5×10⁴. At 10 μg/mlconcentration of antibody, costimulation by Hum231#2 also inducedproliferation of CD4⁺ T cells from 7.5×10⁴ to 10.6×10⁴. Notably,costimulation of CD4⁺ T-cells with 10 μg/ml of m6C8 (InternationalPublication No: WO 06/105021) did not cause additional increases in theabsolute number of cells after 5 days of culture over that seen withanti-CD3/anti-CD28 stimulation alone.

6.3.2 Effect of Agonistic Anti-GITR Antibodies on Anti-CD3 Induced CD4+T-Cell Cytokine Production

As further evidence for the agonistic costimulatory activity ofanti-GITR antibodies, cytokines (IFN_(γ), L-6, TNFα, and IL-10) releasedby CD4+ T cells were measured by multiplex ELISA (Flowcytomix, FACS beadbased cytokine ELISA, eBioscience). Supernatants harvested fromproliferation assays were collected and used for cytokine analysis.FIGS. 10A to 10D show the effect of 10 μg/ml or 5 μg/ml of chimericparental 231-32-15, Hum231#1 or Hum231#2 anti-GITR antibodies oncytokine production by human CD4⁺ T cells. The addition of either 10μg/ml or 5 μg/ml of chimeric parental 231-32-15 antibody, Hum231#1 orHum231#2 to anti-CD3/anti-CD28 stimulated T-cells significantlyincreased the production of IFN_(γ), TNFα, IL-10 and IL-6 when comparedto anti-CD3/anti-CD28 stimulus alone. No costimulatory activity wasobserved for agonistic anti-GITR antibodies in the absence ofanti-CD3/anti-CD28 stimulus.

6.3.3 Titration of Humanized 231-32-15 Clones

To evaluate the range of anti-GITR antibody concentrations that inducesT-cell proliferation and cytokine production, enriched CD4⁺ T-cells werestimulated with 125 ng/ml anti-CD3/anti-CD28 and costimulated withtitrated, plate bound chimeric parental 231-32-15, Hum231#1 or Hum231#2anti-GITR antibodies. The result shown in FIG. 11 indicates thatchimeric parental 231-32-15, Hum231#1 and Hum231#2 costimulation at aconcentration of 10 μg/ml, 5 μg/ml or 2.5 μg/ml induces T cellproliferation as monitored by CFSE dilution. Moreover, in the absence ofany anti-CD3/anti-CD28 stimulus, anti-GITR antibodies do not stimulateCD4⁺ T cell proliferation.

FIG. 12A shows that anti-CD3/anti-CD28 stimulation and costimulationwith anti-GITR antibodies (chimeric parental 231-32-15, Hum231#1 orHum231#2) over a range of concentrations (10 μg/ml, 5 μg/ml or 2.5μg/ml) enhanced CD4⁺ T-cell production of IFN_(γ). Notably, in theabsence of anti-CD3/anti-CD28 stimulation, anti-GITR antibodies(chimeric parental 231-32-15, Hum231#1 or Hum231#2) did not induceIFN_(γ) production.

To examine further the functional activity of chimeric parental231-32-15, Hum231#1 or Hum231#2 anti-GITR antibodies in solution,enriched CD4⁺ T cells were stimulated with 125 ng/ml anti-CD3/anti-CD28and costimulated with titrated soluble anti-GITR antibodies. SolubleHum231#1 or Hum231#2 anti-GITR antibodies also costimulated CD4⁺ T cellsproduction of IFN_(γ), as shown in FIG. 12B.

6.3.4 Effect of Agonistic Anti-GITR Antibody on Anti-CD3 Induced PBMCCytokine Production

In this example, cytokine production induced by co-stimulation with theanti-GITR antibody Hum231#2 was examined using PBMCs. PBMCs isolated viaficoll gradient from healthy donor buffy coats (Research BloodComponents, LLC) were stored in liquid nitrogen and thawed on the day ofthe experiment. The cells were resuspended in cell culture media(RPMI+10% FBS+20 U/ml of IL-2) and added to 96-well culture plates thatcontained plate-bound anti-CD3 antibody at various suboptimalconcentrations (0.3-5 μg/ml) and 5 μg/ml of plate-bound anti-GITRantibody or an isotype control IgG1 antibody. The samples were incubatedfor 4 days at 37° C. and 5% CO₂ and cell culture supernatants werecollected on day 2 and day 4. The samples were tested using the V-PLEXProinflammatory Panel1 (human) Kit (Meso Scale Discovery) to measuresecreted cytokines (IFNγ, IL-2, TNFα, IL-10, IL-13 and IL-4) accordingto the manufacturer's instructions.

As depicted in FIG. 13, co-stimulation with the plate-bound anti-GITRantibody Hum231#2 induced secretion of multiple cytokines in PBMCs fromtwo different donors.

6.3.5 Effect of Agonistic Anti-GITR Antibodies on Cytokine ProductionMeasured by Intracellular Cytokine Staining

The agonistic activity of Hum231#2 on cytokine production was furtheranalyzed by intracellular cytokine staining. PBMCs isolated via ficollgradient from healthy donor buffy coats (Research Blood Components, LLC)were stored in liquid nitrogen and thawed on the day of the experiment.The cells were resuspended in cell culture media (RPMI+10% FBS+20 U/mlof IL-2) and added to 96-well culture plates that contained plate-boundanti-CD3 antibody at various suboptimal concentrations (0.3-5 μg/ml) and5 μg/ml of plate-bound anti-GITR antibody or an isotype control IgG1antibody. The samples were incubated for 3-4 days at 37° C. and 5% CO₂.After activation, to inhibit intracellular protein transport, the cellswere treated with Brefeldin A (BD Biosciences) according to themanufacturer's instructions and the samples were incubated for 6 hoursat 37° C. and 5% CO₂. After the incubation, the cells were stained witha FITC viability amine dye (Life technologies) to stain for dead cells.After washing with cold FACS buffer (1×PBS+2% FBS, pH7.2), an antibodycocktail containing antibodies against CD3 (APC Cy7, SP34.2), CD4 (PercPCy5.5, L200) and CD8α (PE Cy7, SK1) diluted in cold FACS buffer wasadded to each sample and incubated for 10 minutes at 4° C. The cellswere fixed and permeabilized with Cytofix-Cytoperm (BD Biosciences) forintracellular staining according to the manufacturer's instructions. ThePBMCs were stained with antibodies against IFNγ (Alexa647, B27) and TNFα(PE, Mab11) and incubated at room temperature for 10 minutes. Thesamples were washed using 1×Perm-wash buffer (BD Biosciences) andacquired using a FACScanto flow cytometer (BD Biosciences). Flowcytometry plots were analyzed using Flojo software. The flow cytometryplots and graphs are representative of experiments using PBMCs from sixdifferent donors.

The GITR antibody Hum231#2 demonstrated co-stimulatory activity on humanT cells, inducing IFNγ+ monofunctional T cells, TNFα+ monofunctional Tcells as well as IFNγ+ TNFα+ polyfunctional T cells over a range ofsuboptimal anti-CD3 antibody concentrations (FIGS. 14A and 14B).

Next, Hum231#2w, which is a human IgG1 antibody, was converted to ahuman IgG4 antibody named pab1989. The antibody pab1989 shares the sameheavy chain variable region and the same light chain as Hum231#2w butcomprises a human IgG4 constant region. The antibody pab1989 comprises aheavy chain sequence of SEQ ID NO: 554 and a light chain sequence of SEQID NO: 576.

The IgG4 antibody pab1989 was tested in parallel with the IgG1 antibodyHum231#2w in the intracellular cytokine staining experiment describedabove. The anti-CD3 antibody was used at 0.7, 0.8 and 0.9 μg/ml and theanti-GITR antibodies at 5 μg/ml. The samples were incubated for 3-4 daysat 37° C. and 5% CO₂. As shown in FIG. 14C, pab1989 exhibited similaragonistic activity as Hum231#2w, inducing IFNγ+ TNFα+ polyfunctionalCD4+ T cells and TNFα+ monofunctional CD4+ T cells. The graphs arerepresentative of experiments using PBMCs from four different donors.

6.3.6 Effect of Cross-Linking on the Agonistic Activity of Anti-GITRAntibody

The effect of cross-linking on the functional activity of the anti-GITRantibody Hum231#2 was examined using anti-CD3 stimulated PBMCs.

Plate-bound or soluble Hum231#2 was tested for the induction of IFNγ+TNFα+ polyfunctional T cells in a suboptimal CD3 stimulation assay asdescribed in Section 6.3.5. As shown in FIG. 15A, only plate-bound, butnot soluble, Hum231#2 increased the percentage of IFNγ+ TNFα+polyfunctional CD8+ T cells, as compared with the isotype control.

The PBMC cytokine secretion assay as described in Section 6.3.4 wasrepeated for plate-bound Hum231#2 or Hum231#2 cross-linked with ananti-Fc antibody. The culture supernatant was collected on day 4 formeasuring secreted cytokines (IFNγ, IL-2, TNFα, IL-10, IL-13 and IL-4).Co-stimulation with plate-bound (FIG. 15B) or anti-Fc cross-linked (FIG.15C) Hum231#2 induced cytokine secretion.

6.3.7 Activity of Humanized 231-32-15 Clones on Buffy Coat 8 (BC8) andMeasurement of Effector T-Cells or T Regulatory Cells

In this example, the effects of agonistic anti-GITR antibodies on CD4⁺ Teffector or CD4⁺ T regulatory cells were measured by monitoring theirproliferation. Enriched CD4⁺ T-cells were labeled with CFSE and werestimulated with 125 ng/ml plate bound anti-CD3 antibody. Proliferatedenriched CD4⁺ T-cells were monitored by CFSE dilution after 5 days inculture.

The CD4⁺ T effector or T regulatory cell population within the enrichedCD4⁺ T cells population shown in FIGS. 16A and 16B was identified byflow cytometry staining by their cell surface markers. Activated CD4⁻ Teffector cells were characterized as CD25⁺, CD45RA⁻, CD127^(Med/Low) andFoxp3^(Neg/Low). CD4⁺ T regulatory cells were identified as CD4⁺, CD25⁺,CD45RA−, CD127^(Low), and Foxp3^(High). FACS staining was performedaccording to Table 14 below:

TABLE 14 FACS staining panel Antigen/ Channel Laser Standard FlurochromeFluorochrome 1 Blue 488 nm FITC, AF488, GFP CFSE 2 Blue 488 nm PE CD1276 Blue 488 nm Pe-Cy7 CD45RA 7 Red 633 nm APC GITR APC 9 Red 633 nmAPC-Cy-7, APC-H7 CD25-APC-H7 10 Violet 405 nm DAPI, Pac Blue, V450 FoxP3e450 11 Violet 405 nm AF430, AmCyan, V500 L/D

Results of the stimulation assay are shown in the FACS plots of FIGS.16A and 16B. Gating on CD4⁺ Tregs (CD4⁺, CD25⁺, CD45RA⁻, CD127^(Low) andFoxp3^(High)) or activated CD4⁺ T effector cells (CD25⁺, CD45RA⁻,CD127^(Med/Low) and Foxp3^(Neg/Low)) shows that 125 ng/mlanti-CD3/anti-CD28 stimulation alone and in conjunction with anti-GITRcostimulation upregulated GITR expression on both T effector and Tregulatory cells.

FIG. 16A depicts FACS analysis of both T-effector and T-regulatorycells. Both cell types expressed GITR on their cell surface followingstimulation with anti-CD3 alone or in conjunction with anti-GITRantibodies. However, co-stimulation with anti-GITR antibodiespreferentially expands T-effector cells over T-regulatory cells, leadingto an increased Teff/Treg ratio (FIG. 16B).

As further evidence for the agonistic activity of anti-GITR antibodieson T-cells in the context of cellular immunity, T cell responsesfollowing stimulation of PBMCs were evaluated.

Stimulation of PBMC was titrated by adjusting anti-CD3-inducedproliferation on PBMCs. Illustrated in FIGS. 17A and 17B areCFSE-labeled PBMCs stimulated with 31.25 ng/ml of plate boundanti-CD3/anti-CD28 in conjunction with either plate bound anti-GITRantibodies or isotype control. As a positive control for the activity ofanti-GITR antibodies, the same stimulation condition was used tostimulate enriched CD4⁺ T cells (data not shown).

CD4⁺ or CD8⁺ T-cells in the PBMC population were identified by theirstaining with anti-CD3 and anti-CD4 or anti-CD3 and anti-CD8. FloJo(Tree Star, Inc.) analysis of acquired FACS samples gated on CD4⁺CD3⁺ Tcells or CD8⁺CD3⁺ T cells showed anti-GITR chimeric parental 231-32-15(REF 231), Hum231#1 and Hum231#2 antibodies stimulated T cellproliferation (% CFSE low). In particular, the experiment depicted inFIG. 17B revealed that anti-GITR chimeric parental 231-32-15 (REF 231),Hum231#1 and Hum231#2 antibodies have activity on CD8⁺ T cells.

6.3.8 Effect of Agonistic Anti-GITR Antibodies on GITR NF-κB-LuciferaseReporter Cell Line

A human GITR NF-κB-luciferase reporter cell line (Promega) was designedto probe the co-stimulatory activity of anti-GITR agonistic antibodies.Activation of GITR by anti-GITR agonistic antibody or GITR ligand hasbeen reported to activate NF-κB (Snell L M et al., (2010) J Immunol 185:7223-7234; Bulliard Y et al., (2013) J Exp Med 210: 1685-1693; Yu K Y etal., (2003) Biochem Biophys Res Commun 310: 433-438). As such, Jurkatcells were genetically modified to stably express the GloResponseNF-κB-luc2P construct and human GITR. The reporter cells wereresuspended in assay media (RPMI+1% FBS) and incubated with variousconcentrations (12.5, 10, 5, 2.5, 1.25, and 0.625 μg/ml) of plate-boundanti-GITR antibodies Hum231#2w, m6C8 or an IgG1 isotype control in theabsence or presence of 0.3 μg/ml of a plate-bound anti-CD3 antibody(Clone SP34). The plates incubated with the anti-CD3 antibody were readafter 6 or 18 hours of incubation. The plates without the anti-CD3antibody were read after 2, 5, 6, 8 or 18 hours of incubation. Afterincubation, the plates were equilibrated at room temperature and then anequal volume of room temperature Bio-Glo reagent (Promega) was added.Luminescence was read using an EnVision multilabel reader 2100.

For the assay with the anti-CD3 antibody, the luciferase RLU at 18-hourpost-stimulation was plotted for each antibody concentration tested(FIG. 18A). Similarly, for the assay without the anti-CD3 antibody, FIG.18B is a graph showing the luciferase relative light units (RLU) at5-hour post-stimulation for various antibody concentrations tested. InFIG. 18C, the highest ratios of luciferase expression (GITR Ab/isotypecontrol) without the anti-CD3 antibody, among several antibodyconcentrations tested, at 0, 2, 5, 6, 8 and 18 hours post-stimulationare shown. The data shown are representative of four experiments withthe anti-CD3 antibody or two experiments without the anti-CD3 antibody.

In the presence of the anti-CD3 antibody, although m6C8 showed strongeragonistic activity at 6 hour (data not shown), by 18-hourpost-stimulation, Hum231#2w and m6C8 induced similar activation of theGITR reporter cell line (FIG. 18A). However, in the absence of theanti-CD3 antibody, only Hum231#2w but not m6C8 induced activation of theGITR reporter cell line (FIG. 18B).

6.3.9 Effect of Agonistic Anti-GITR Antibody on Fc Gamma Receptor IIIA(CD16) Reporter Cell Line

In this example, expression of human GITR by activated nTreg cells and Teffector cells was examined. PBMCs isolated from healthy donors wereenriched for CD3+ T cells (Teff) or CD4+CD25+CD45RA+ T cells (nTregs)using magnetic-based separation techniques. T lymphocytes were thenactivated with CD3-CD28 expansion beads with 500 U rIL-2 for 4 days, and50 U rIL-2 for an additional 5 days. GITR receptor quantitation wasdetermined by flow cytometry by gating on CD4+ and CD8+ Teff versusnTreg. Quantibrite beads (BD Biosciences) were run simultaneously andused to quantify GITR receptor surface density.

As shown in FIG. 19A, the surface expression of human GITR on activatednTreg cells at day 9 (and all time points evaluated) was higher thanthat on activated CD4+ or CD8+ T effector cells.

Next, the ability of anti-GITR antibody Hum231#2w to co-engage GITR andsignal via activating Fc gamma receptors was evaluated using a reportercell line expressing Fc gamma receptor IIIA (CD16) together withactivated T effector (Teff) or nTreg cells, generated as described.Expanded Teff or nTreg cells were incubated with different doses ofHum231#2w or an IgG1 isotype control. Jurkat NFAT-luciferase reportercells overexpressing CD16 (158 V/V polymorphism) were added to thesamples. Binding of the antibody/antigen complex, wherein the antigen islocated on the cell surface, to CD16 signals to the promoter/reporterconstruct and results in luciferase gene transcription. Plates wereincubated for 20 hours at 37° C. and 5% CO₂. After this incubation,Bio-Glo Luciferase Assay Reagent (Promega) was thawed at roomtemperature and 75 μl was added to each well of the 96-well white assayplates. Within 5-10 minutes, luminescence was measured. Backgroundluminescence was subtracted from each sample reading and the adjustedrelative light units (RLU) were recorded. ΔRLU represents the RLU of theanti-GITR antibody minus that of the isotype control.

Consistent with the differential surface GITR expression betweenactivated nTregs and activated CD4+ or CD8+ T effector cells (FIG. 19A),the anti-GITR antibody Hum231#2w preferentially activated CD16 whenbound to activated nTreg cells (FIG. 19B).

To evaluate if GITR overexpression was a feature of regulatory T cellslocated within tumor microenvironment, GITR expression was compared on Tcells isolated from the blood of healthy human donors (FIG. 19C, a-c,n=3) or from tumor tissues of non-small cell lung cancer (NSCLC)patients (FIG. 19C, d-f, n=3). To eliminate background binding ofantibodies to immune populations, all the cells were incubated withpurified CD16/32 antibody (10 μg/ml, 20 minutes at room temperature)prior to the addition of cell-surface and intracellular antibodies.Following FcR-blockade, all the samples were incubated withAPC-conjugated anti-GITR antibody (clone 110416, R&D systems) or isotypecontrol and a cell-surface antibody lineage-cocktail (CD3-FITC,CD25-PECy7, CD4-BV650 and CD8a-PE) for 45 minutes on ice (1 μg/ml each),washed three times with FACS buffer (PBS, EDTA and 0.5% BSA), followedby fixation/permeabilization and incubation with Pacific Blue-conjugatedFOXP3 (fix/perm and incubation each 45 minutes on ice, 1 μg/ml). Thestained samples were then analyzed using a LSRFortessa flow cytometer(BD Biosciences). The cell populations in FIG. 19C were defined as:Tconv (CD3+, CD4+, CD8a−, CD25low, FOXP3−) or Treg (CD3+, CD4+, CD8a−,CD25high, FOXP3+).

As demonstrated in FIG. 19C, GITR surface expression was highest onregulatory T cells isolated from the tumor tissues of NSCLC patients,with little or no detectable level on Treg or conventional T cells fromhealthy donors.

6.3.10 Effect of Agonistic Anti-GITR Antibody on the Cytokine Productionof T Cells from African Green Monkey

To test for species cross-reactivity, Hum231#2 was evaluated for itsbinding to GITR from African green monkey (AGM). Briefly, AGM PBMCs(Worldwide Primates) were thawed and counted. The PBMCs were resuspendedin cell culture media (RPMI+10% FBS) and stimulated with an anti-CD3antibody (clone SP34.2, BD) or ConA (Sigma) plus IL-2 (20 U/ml) for 3days at 37° C. and 5% CO2. Following activation, the cells were stainedwith amine dye FITC (Life technologies) for 15 minutes at roomtemperature. The cells were washed with cold FACS buffer (1×PBS+2% FBS,pH7.2) and an antibody cocktail diluted in cold FACS buffer containingantibodies against CD3 (APC Cy7, SP34.2), CD4 (PercP, L200), CD8 (PECy7, SK1) and PD-1 (PE, EH12.2H7) was added and incubated for 10 minutesat 4° C. The cells were washed and incubated with 2.5 μg per well ofHum231#2 or IgG1 isotype control for 10 minutes at 4° C. The cells werewashed and then stained with a secondary anti-Fc F(ab′)₂ antibodyconjugated with Alexa647 for 10 minutes at 4° C. The cells were washedand fixed with 1.6% paraformaldehyde before acquisition using aFACSCanto flow cytometer (BD Biosciences). FACS files were analyzedusing FACS DIVA software.

As shown in FIG. 20A, the anti-GITR antibody Hum231#2 binds to activatedAGM CD4+ and CD8+ T cells. Unstimulated T cells from AGM do not expressbaseline levels of GITR and the GITR levels on the cell surface areup-regulated upon T cell activation. The plots shown in FIG. 20A arerepresentative of experiments using PBMCs from three different AGMs.

Next, a CD3 substimulation assay was performed using PBMCs from Africangreen monkey (AGM) to examine the agonistic activity of Hum231#2w. Human(Research Blood Components, LLC) or AGM PBMCs (Worldwide Primates) wereisolated via ficoll gradient from healthy donors and were stored inliquid nitrogen and thawed on the day of the experiment. The cells wereresuspended in cell culture media (RPMI+10% FBS+20 U/ml of IL-2) andadded to 96-well culture plates that contained plate-bound anti-CD3antibody (0.8 μg/ml) and various concentrations (2, 4, 5, 6 and 9 μg/ml)of plate-bound anti-GITR antibody or an isotype control IgG1 antibody.The samples were incubated for 4 days at 37° C. and 5% CO₂. Afteractivation, to inhibit intracellular protein transport, the cells weretreated with Brefeldin A (BD Biosciences) according to themanufacturer's instructions and the samples were incubated for 6 hoursat 37° C. and 5% CO₂. After the incubation, the cells were stained witha FITC viability amine dye (Life technologies) to stain for dead cells.After washing with cold FACS buffer (1×PBS+2% FBS, pH7.2), an antibodycocktail containing antibodies against CD3 (APC Cy7, SP34.2), CD4 (PercPCy5.5, L200) and CD8α (PE Cy7, SK1) diluted in cold FACS buffer wasadded to each sample and incubated for 10 minutes at 4° C. The cellswere fixed and permeabilized with Cytofix-Cytoperm (BD Biosciences) forintracellular staining according to the manufacturer's instructions. ThePBMCs were stained with antibodies against IFNγ (Alexa647, B27) and TNFα(PE, Mab11, only for human PBMCs) and incubated at room temperature for10 minutes. Samples were washed using 1×Perm-wash buffer (BDBiosciences) and acquired using a FACScanto flow cytometer (BDBiosciences). Flow cytometry plots were analyzed using Flojo software.

As shown in FIGS. 20B and 20C, co-stimulation with the anti-GITRantibody Hum231#2w induces IFNγ production by CD8+ AGM T cells. The flowcytometry plots and graphs are representative of experiments using PBMCsfrom two AGMs.

6.3.11 Effect of the Simultaneous Binding of Recombinant Human GITRLigand and Humanized 231-32-15 Clones on Anti-CD3 Stimulated CD4+ TCells

An agonistic anti-GITR antibody which does not prevent GITR binding toGITR ligand (GITRL) may result in an enhanced immune response,characterized by enhancing the proliferation and/or the effectorfunction of T effector cells and/or down-regulating the suppressivefunction of T regulatory cells.

Anti-GITR antibodies, either alone or in combination with recombinanthuman GITRL, are tested for their agonistic activity on CD4⁺ T cells.Enriched CD4⁺ T cells are labeled with 1-2 μM CFSE, washed and thenstimulated with plate bound anti-CD3 (125 ng/ml), soluble anti-CD28 (125ng/ml) and 10 U IL-2, together with 10 μg/ml chimeric parental 231-32-15antibody, 10 μg/ml Hum231#1, 10 μg/ml Hum231#2, 10 μg/ml GITRL, acombination of 10 μg/ml chimeric parental 231-32-15 antibody with 10μg/ml GITRL, a combination of 10 μg/ml Hum231#1 with 10 μg/ml GITRL, ora combination of 10 μg/ml Hum231#2 with 10 μg/ml GITRL, at 37° C. for 3to 6 days. The culture supernatants and cells are then collected fromthe plates. Cell proliferation and cytokine release are examined asdescribed in Sections 6.3.1 and 6.3.2, respectively. The effects of thesimultaneous binding of anti-GITR antibodies and GITRL on CD4⁺ Teffector cells or CD4⁺ T regulatory cells can be further tested asdescribed in Section 6.3.7. This study may show a synergistic oradditive effect between GITRL and anti-GITR antibodies (chimericparental 231-32-15, Hum231#1 and Hum231#2) in enhancing immuneresponses.

As an alternative to using soluble recombinant human GITRL for testingco-agonist activity in combination with the anti-GITR antibodiesdescribed herein, it is also possible to use antigen-presenting cellsthat are induced to express GITRL. Such induced APCs may be culturedwith CD4⁺ T effector cells or CD4⁺ T regulatory cells, as describedabove, in the presence or absence of the anti-GITR antibodies and thefunction of the T cells assessed. To induce GITRL expression, antigenpresenting cells such as macrophage or dendritic cells are incubatedwith a TLR4 ligand (e.g., LPS) for 1, 2, 4, 6 or 12 hours as described,e.g., in Tone M et al., (2003) PNAS 100: 15059-15064; or with wholeβ-glycan particles (WGP) purified from the cell wall of Saccharomycescerevisiae for 6, 12, 24, 48 or 72 hours, as described, e.g., in Tian Jet al., (2012) PLoS One, 7(10): e46936.

6.3.12 Effect of Agonistic Anti-GITR Antibody on OX40 and PD-1 SurfaceExpression on T Cells

In this example, the agonistic anti-GITR antibody was evaluated for itsimpact on the surface expression of OX40 and PD-1 on T cells. PBMCsisolated via ficoll gradient from healthy donor buffy coats (ResearchBlood Components, LLC) were stored in liquid nitrogen and thawed on theday of the experiment. The cells were resuspended in cell culture media(RPMI+10% FBS+20 U/ml of L-2) and added to 96-well culture plates thatcontained plate-bound anti-CD3 antibody (clone SP34) at varioussuboptimal concentrations (0, 0.7, 0.8 and 0.9 μg/ml) and g/ml of theplate-bound anti-GITR antibody Hum231#2 or an isotype control IgG1antibody. The samples were incubated for 4 days at 37° C. and 5% CO₂.After the incubation, the cells were stained with a FITC viability aminedye (Life technologies) to stain for dead cells. After washing with coldFACS buffer (1×PBS+2% FBS, pH7.2), an anti-OX40 antibody was added andincubated for 10 minutes at 4° C. The cells were washed and ananti-human Fc F(ab′)₂ Alexa647 was added and incubated for 10 minutes at4° C. After centrifugation and a washing step, an antibody cocktailcontaining antibodies against CD3 (APC Cy7, SP34.2), CD4 (PercP Cy5.5,L200), CD8α (PE Cy7, SK1) and PD-1 (PE, EH12.2H7) diluted in cold FACSbuffer was added to each sample and incubated for 10 minutes at 4° C.The samples were washed and resuspended in 200 μl of 1.6%paraformaldehyde before acquisition using a FACScanto flow cytometer (BDBiosciences). FACS plots were analyzed using Flojo software. The flowcytometry plots and graphs are representative of experiments using PBMCsfrom one donor.

As shown in FIG. 21, co-stimulation with the anti-GITR antibody Hum231#2increases OX40 and PD-1 surface expression on human CD4+ and CD8+ Tcells.

6.4 Example 4: Germlining of Humanized Variant

This example describes the generation of germline, humanized variants.

6.4.1 Library Design

A library approach was used to generate humanized variants with anincreased human germline content by introducing site directed mutationsvia degenerate codons into heavy and light chain variable regions. Thevariable region of the VH chain was mutated by replacing 17 amino acidspositions with 2 to 4 amino acids, resulting in a final diversity of1.3E+06. The variable region of light chain was mutated at 9 amino acidpositions (2-3 amino acids per position), resulting in a final diversityof 7.7E+02. The different framework and CDR positions included in thelibrary are shown in FIG. 22. The libraries were designed usingIGHV1-2*02 VH human germline (FIG. 22A) and IGKV4-1*01 VL human germline(FIG. 22B).

6.4.2 Library Generation

The mutated humanized variable regions were cloned into retroviralexpression vectors (pCMA). These constructs were subsequently used totransduce preB cells and express antibodies on the surface usingRetrocyte Display® technology. The retroviral expression vectorcontained MSCV based 5′ and 3′LTR's, immunoglobulin constant region(IGHG1 or IGKC) comprising membrane anchor fraction (IGHG1) and a CD4surface marker gene. The surface marker and immunoglobulin are coupledby IRES (internal ribosome entry site). The term “variable region” inthis example means VDJ rearranged genes for the heavy chain and VJrearranged genes for the light chains.

6.4.2.1 Generation of a Humanized Heavy Chain Library

Synthesised humanized variable heavy chain regions (Eurofins MWG GmbH)were cloned into the retro-viral expression vector containingimmunoglobulin constant region (IGHG1) comprising membrane anchorfraction. Digestion and ligation were performed in one step and one tubeusing the typeIIS restriction enzyme LguI and T4-DNA Ligase at 37° C.for 1 hour. The synthesized humanized heavy chain library material(128.7 ng) was ligated in frame into a pCMA retroviral expression vector(1 μg) at a vector to insert ratio of 1:3. The ligation reaction wasthen precipitated and concentrated (8.3-fold) to a final DNAconcentration of 94 ng/μl.

The whole (3×4 μl) concentrated ligation reaction was electroporatedinto 80 μl of DH10B cells (E. coli ElectroMax DH10B electrocompetentcells, Invitrogen, Cat No 12033-015) (1900V/5 ms). 1000 μl SOC medium(Invitrogen, Cat No 15544-034) was added and the transformed DH10B cellsrecovered at 37° C. for 1 hour. A 1:1000 dilution was performed todetermine the library complexity. The whole transformation reactionswere plated onto LB-agar +100 μg/ml ampicillin plates and incubated overnight at 37° C. The complexity of humanized VH chain library wasdetermined to be 7.3E+07 and therefore the whole library diversity wasrecovered.

All electroporated bacteria were scratched from the plates and 2glycerol stocks for long-term storage at −80° C. were prepared. Alarge-scale DNA plasmid preparation (Macherey & Nagel, NucleoBond XtraMaxi Plus Kit) was performed. A digestion to verify the presence and thecorrect size of cloned insert humanized VH chain library material wasperformed with HindIII/Eco47III (H/E). To verify the correct vectorbackbone a KpnI/BsrGI (K/B) digestion was used. As a control thehumanized VH chain library plasmid DNA was also digested with LguI toverify the amounts of the vector without insertions of humanized VHchains and the vector integrity was tested by separation of uncutplasmid DNA.

96 single clones were picked and sent for sequencing to determine thefinal library diversity using primer 89-AL (Sequence: 5′gcctccgcctcctcttcctccatcc 3′; SEQ ID NO: 707). No redundant sequenceswere identified in quality control. The theoretical diversity is 1.3E+06different variants hence the coverage of each unique sequence is about50 times. 35% of the library-clones had the desired mutation pattern(=2.5E+07) and all desired variants were present in the library.

6.4.2.2 Generation of a Humanized Light Chain Library

Synthesised humanized variable light chain regions (Eurofins MWG GmbH)were cloned into the retroviral expression vector containingimmunoglobulin constant region (IGKC). Digestion and ligation wereperformed as described in section 6.4.2.1. The synthesized humanizedlight chain library material (227.9 ng) was ligated in frame into a pCMAretro-viral expression vector (0.5 μg) at a vector to insert ratio of1:10. Afterwards the ligation reaction was precipitated and 3.6-foldconcentrated to a final DNA concentration of 52 ng/μl.

Transformation was performed as described in Section 6.4.2.1 above. 2×4μl concentrated ligation reaction was electroporated into 80 μl of DH10Bcells (E. coli ElectroMax DH10B electrocompetent cells, Invitrogen, CatNo 12033-015) (1900V/5 ms). The complexity of the humanized VL chainlibrary was determined to be 4.6E+07 and therefore the whole librarydiversity was recovered. The library plasmid DNA preparation and theverification of plasmid DNA was performed as described in Section6.4.2.1. Only one redundant sequence was identified in quality control.The theoretical diversity is 7.7E+02 different variants hence thecoverage of each unique sequence is about 60000 times. 65% of thelibrary-clones had the desired mutation pattern and all desired variantswere present in the library.

6.4.3 Recovery of Germlined Heavy and Light Chains from Pre-SelectedpreB Cell Clones

The humanized library material generated as described above (Sections6.4.2.1 and 6.4.2.2) was used in an affinity maturation RetrocyteDisplay® screen to identify antibodies with a high germline gene contentand improved biological and biochemical properties. From two 96 wellplates, containing 80 and 96 pre-selected preB cell clones, heavy andlight chains were recovered. The cells were lysed (Phusion HumanSpecimen Direct PCR Kit, Thermo Scientific/Finnzymes Cat. No. F-150) andthe variable regions were amplified directly by PCR (see Table 15). ThePCR was performed with a specific 5′ forward and 3′ reverse primer (seeTable 16). As a template for the PCR 2 μl preB cell lysate was used.Amplified variable regions were purified (NucleoFast 96 PCR(Macherey-Nagel)) and cloned into the CHO expression vectors (pPEP)containing immunoglobulin constant region (IGHG1, IGKC).

TABLE 15 PCR Programs  1. Initial denaturation 98° C. 5 min 34 cycles 2. Denaturation 98° C. 1 s  3. Annealing/Elongation 72° C. 15 s 11.Final elongation 72° C. 1 min 12. Cooling 10° C. Hold Total no of cycles35

TABLE 16Primers to amplify the heavy and kappa light chain variable regions NameSequence SEQ ID NO: PCR forward Primers (5′) 5′ hum231-32-15 Vh 5′tctgctcttctaccatggattggacttggcgcattctgttc 3′ 708 LguI (1192-Je) 5′hum231-32-15 Vk 5′ cttgctcttctatggtgttacagactcaggtgttc 3′ 709LguI (1193-Je) PCR reverse Primers (3′) 3′ H LguI Cg 5′tacgctcttcaagctgctggagggcacgg 3′ 710 (1060-Je) 3′ K LguI Ck 5′cttgctcttcgctcagcgtcagggtgct 3′ 711 (1065-Je)

To clone the pre-selected germlined heavy and light chain variableregions, digestion and ligation were performed in one step and one tubeusing the typeIIS restriction enzyme LguI and T4-DNA Ligase at 37° C.for 1 h and a final step at 80° C. for 10 min, and the pre-selectedgermlined heavy or light chain variable regions (˜60 ng) were ligated inframe into pPEP expression vectors (50 μg). A vector to insert ratio of1:12 was used.

2 μl pre-selected germlined heavy chain ligation reactions and 6 μlpre-selected germlined kappa light chain ligation reactions wereco-transformed in chemical competent DH10B cells (30 μl) by heat shocktransformation. 1000 μl SOC medium (Invitrogen, Cat No 15544-034) wasadded and the transformed DH10B cells recovered at 37° C. for 1 hour.Finally 1000 μl LB-medium+ampicillin (final concentration: 100 μg/ml)was added and the transformed E. coli cells incubated overnight at 37°C. A DNA plasmid preparation in small-scale (Macherey & Nagel,NucleoSpin 96 Plasmid) was carried out and a digestion to verify thepresence and the correct size of cloned variable regions was performedwith HindIII/NotI (VH chains) and NcoI (VK chains). The vector integritywas tested by separation of uncut plasmid DNA. The DNA plasmidpreparations were subsequently used to transfect CHO cells and theexpressed antibodies were tested using suspension array technology andin kinetic analysis by Octet. Antibody sequences were verified by PCR.

6.4.4 Selection of Germline Variants

Several hundred germlined antibodies were selected based on bindingkinetics as determined by Octet measurements (Octet RED 96 system;ForteBio™ Inc., Menlo Park, Calif.). The experimental procedure wasconfigured according to the instruction manual of the instrument.Biotin-GITR was bound to Streptavidin biosensor (SA) and PBS (pH 7.4)was used as a blank control. Briefly, the interaction analyses wereconducted at 30° C. in running buffer (PBS, 0.05% Tween, pH7.4). Sensortips were pre-wet for 10 minutes in running buffer immediately prior touse, and the micro plates used in the Octet were filled with 200 μl ofsample or buffer per well and agitated at 800 rpm. For the experiments,commercially available precoated SA tips were used. Biotinylated GITRwas loaded onto the ForteBio SA fibers in PBS pH7.4 for 10 minutes andwashed for 4 minutes. For the association phase, the ligand-coated SAtips were immersed for 5 minutes in cell culture supernatant that wasdiluted 1:10 in running buffer prior to the measurement. Dissociation ofthe antibody-antigen complex was measured in wells containing the Octetbuffer alone for 5 minutes. After each run, the tips were regeneratedwith Glycine (10 mM, pH 2.0). Affinity, K_(on) and K_(off) weredetermined with Octet evaluation software v6.3 using a 1:1 binding modelwith local full fitting. Table 17 lists the composition of the heavychain variable regions and light chain variable regions of 56 selectgermline variants and their affinity, K_(on) and K_(off) values asmeasured by Octet.

TABLE 17 Kinetic analysis of germline variants Heavy chain Light chainAntibody variable region variable region Affinity k_(on) K_(off) ID (SEQID NO:) (SEQ ID NO:) (M) (1/Ms) (1/s) 1 H1916A01 (215) K1916A01 (400)7.44E−10 4.68E+05 3.48E−04 2 H1916A03 (217) K1916A03 (401) 4.23E−095.53E+05 2.34E−03 4 H1916A05 (219) K1916A05 (403) 1.43E−09 3.51E+055.01E−04 5 H1916A06 (220) K1916A06 (404) 1.63E−09 5.91E+05 9.66E−04 6H1916A07 (221) K1916A07 (405) 2.70E−09 2.14E+05 5.79E−04 9 H1916A10(224) K1916A10 (408) 2.68E−09 2.93E+05 7.84E−04 10 H1916A11 (225)K1916A11 (409) 9.11E−10 6.15E+05 5.60E−04 11 H1916A12 (226) K1916A12(410) 2.10E−09 4.03E+05 8.47E−04 15 H1916B05 (230) K1916B05 (415)2.22E−09 2.78E+05 6.16E−04 16 H1916B06 (231) K1916B06 (416) 1.47E−093.56E+05 5.23E−04 18 H1916B09 (233) K1916B09 (419) 3.77E−09 2.19E+058.24E−04 20 H1916B12 (236) K1916B12 (421) 1.35E−09 2.83E+05 3.80E−04 21H1916C03 (237) K1916C03 (423) 8.44E−09 3.09E+05 2.61E−03 25 H1916C07(241) K1916C07 (427) 1.69E−09 3.74E+05 6.32E−04 29 H1916C11 (245)K1916C11 (431) 1.06E−09 2.95E+05 3.13E−04 31 H1916D01 (247) K1916D01(433) 4.18E−10 5.05E+05 2.11E−04 33 H1916D03 (249) K1916D03 (435)9.01E−11 1.12E+07 1.01E−03 34 H1916D04 (250) K1916D04 (436) 4.58E−105.57E+05 2.55E−04 35 H1916D05 (251) K1916D05 (437) 1.87E−10 1.29E+062.40E−04 36 H1916D06 (252) K1916D06 (438) 4.40E−10 6.38E+05 2.80E−04 37H1916D07 (253) K1916D07 (439) 3.17E−11 7.64E+06 2.42E−04 38 H1916D08(254) K1916D08 (440) 8.75E−11 8.57E+06 7.50E−04 39 H1916D09 (255)K1916D09 (441) 2.55E−10 3.91E+06 9.97E−04 42 H1916E01 (259) K1916E01(444) 3.77E−10 4.17E+05 1.57E−04 43 H1916E03 (261) K1916E03 (445)1.28E−09 3.73E+05 4.77E−04 45 H1916E05 (263) K1916E05 (447) 5.62E−104.55E+05 2.55E−04 46 H1916E06 (264) K1916E06 (448) 6.19E−10 4.00E+052.48E−04 47 H1916E08 (265) K1916E08 (450) 2.06E−09 3.91E+05 8.04E−04 49H1916E11 (268) K1916E11 (452) 2.09E−09 3.38E+05 7.07E−04 50 H1916F03(270) K1916F03 (454) 1.01E−09 2.52E+05 2.54E−04 52 H1916F05 (272)K1916F05 (456) 1.07E−09 3.97E+05 4.26E−04 54 H1916F09 (276) K1916F09(458) 1.26E−09 5.48E+05 6.88E−04 55 H1916F10 (277) K1916F10 (459)1.27E−09 4.35E+05 5.50E−04 58 H1916G04 (283) K1916G04 (462) 1.58E−092.63E+05 4.15E−04 59 H1916G05 (284) K1916G05 (463) 1.04E−09 2.99E+053.12E−04 61 H1917A02 (287) K1917A02 (467) 1.83E−09 7.72E+05 1.41E−03 68H1917B01 (298) K1917B01 (474) 1.55E−09 2.89E+05 4.47E−04 70 H1917B04(301) K1917B04 (476) 2.01E−09 4.37E+05 8.79E−04 71 H1917B07 (304)K1917B07 (477) 2.41E−10 1.05E+06 2.52E−04 75 H1917C09 (313) K1917C09(484) 2.92E−09 3.17E+05 9.25E−04 76 H1917C10 (314) K1917C10 (485)2.72E−09 3.86E+05 1.05E−03 78 H1917D01 (316) K1917D01 (488) 1.00E−093.25E+05 3.27E−04 79 H1917D04 (319) K1917D04 (489) 2.87E−09 4.50E+051.29E−03 80 H1917D07 (320) K1917D07 (490) 6.96E−10 6.52E+05 4.54E−04 85H1917E02 (327) K1917E02 (495) 1.28E−09 2.89E+05 3.70E−04 86 H1917E03(328) K1917E03 (496) 7.50E−10 5.77E+05 4.32E−04 91 H1917F03 (340)K1917F03 (501) 3.07E−09 5.20E+05 1.59E−03 92 H1917F05 (342) K1917F05(502) 1.01E−09 3.57E+05 3.61E−04 94 H1917G01 (350) K1917G01 (504)1.18E−09 3.72E+05 4.40E−04 95 H1917G05 (354) K1917G05 (505) 2.21E−093.05E+05 6.72E−04 96 H1917G06 (355) K1917G06 (506) 1.09E−09 3.44E+053.73E−04 97 H1917G07 (356) K1917G07 (507) 1.43E−09 5.34E+05 7.61E−04 101H1917H01 (362) K1917H01 (511) 3.54E−10 9.36E+05 3.32E−04 102 H1917H02(363) K1917H02 (512) 1.97E−09 3.21E+05 6.32E−04 105 H1917H07 (366)K1917H07 (516) 1.38E−09 3.51E+05 4.86E−04 107 H1917H09 (368) K1917H09(518) 2.21E−09 2.98E+05 6.57E−04

Of these antibodies, a number were chosen for subsequent analysis basedon further Octet values and their germline homology. From these, asample of antibodies, which were all agonistic in a T cell assay (datanot shown), were characterized in more detail as described below. FIG.23 details the composition of the heavy and light chain variable regionsof these selected germline variants. FIGS. 24A, 24B and 24C detail thecomposition of the heavy and light chain regions of other select actualor prophetic germline variants.

6.4.5 Kinetic Analysis of Germline Variants

Quantification and binding analysis of the anti-GITR germline variantantibodies was determined using suspension array technology followingthe methods as described in Section 6.2.5.1. The mean relativeaffinities of the germline variants compared to the chimeric parental231-32-15 antibody are shown in FIG. 23.

In addition, an assessment of ligand blocking activity using suspensionarray technology was also carried out according to the method describedin Example 6.2.5.2. As can be seen in FIGS. 25A and 25B, GITRL-PEbinding to GITR in the presence of a selection of germline variantantibodies followed a very similar pattern for the antibodies tested.

These germline variant antibodies were further characterized infunctional assays as described below in Example 5.

6.5 Example 5: Functional Activity of Germline Variants 6.5.1 Effect ofGermline Variants on Anti-CD3 Stimulated CD4+ T Cell Proliferation andCytokine Production

In order to assess the activity of new germline variants generated asdescribed in Example 4, these variants were compared to the humanizedantibodies Hum231#1 and Hum231#2 and the chimeric parental 231-32-15antibody on enriched CD4 T cells from four buffy coat preparations, BC4,BC9, BC13 and BC18.

A suboptimal CD3 stimulation assay was performed as described in Section6.3.1 above with intracellular cytokine staining (BC13 and BC18),cytokine release (BC4 and BC9) and CFSE dilution (BC4 and BC9) measured5 days after the stimulation assay was performed. Plate bound anti-CD3and soluble anti-CD28 antibodies were used with plate bound anti-GITRantibodies, no antibody (CD3 alone), and an isotype control (antibodyMSC8). Anti-GITR antibodies were used at a concentration of 10 μg/ml.For buffy coats 4 and 9, anti-CD3 antibody was used at a concentrationof 125 ng/ml, anti-CD28 antibody at a concentration of 125 ng/ml and 10U IL-2. For buffy coat 13, anti-CD3 antibody was used at a concentrationof 500 ng/ml and anti-CD28 antibody at a concentration of 100 ng/ml. Forbuffy coat 18, anti-CD3 antibody was used at a concentration of 31.25ng/ml and anti-CD28 antibody at a concentration of 100 ng/ml.

For the buffy coat preparations BC4 and BC9, the supernatants and cellswere collected from the plate after 5 days in culture. Cellproliferation was determined and is shown as percentage CFSE low (FIGS.26A and 26B) and the supernatants were used for cytokine analysis (IFNγand IL-10). FIGS. 27A and 27B show cytokine release for BC4 and FIGS.28A and 28B show cytokine release for BC9.

For buffy coat preparations BC13 and BC18, following 5 days in culture,monensin (eBioscience) was added to all samples for 6 hours to enableintracellular retention of IFNγ. The samples were then stainedintracellularly for IFNγ-PE (eBioscience) using BD Cytofix/Cytoperm kit(BD Biosciences) following detection by flow cytometry on BD FACSAria I(BD Biosciences) and analysed using FlowJo software (Tree Star). Resultsof the effect of the germlined variants on the percentage of IFNγpositive CD4 T cells from BC13 and BC18 are shown in FIGS. 29A and 29B,respectively. Following contact with the germline variants, the chimericparental 231-32-15 antibody or the humanized variants Hum231#1 andHum231#2, the percentage of IFNγ positive CD4 T-cells induced by theseanti-GITR antibodies were comparable. However, as expected, there issome slight variation between the donors.

6.5.2 Effect of Germline Variants on GITR NF-κB-Luciferase Reporter CellLine

In this example, the germline variants generated as described in Example4 were tested using the GITR NF-κB-luciferase reporter cell line(Promega) described in Section 6.3.8.

The reporter cell line (Promega) was kept in culture according to themanufacturer's instruction. On the day of the experiment, the cells wereresuspended in assay media (RPMI+1% FBS). The cells (100,000 cells perwell) were added to a 96-well plate that contained plate-bound anti-CD3antibody (clone SP34, 0.3 μg/ml) and various concentrations (12.5, 10,5, 2.5, 1.25, 0.625 and 0 μg/ml) of plate-bound anti-GITR antibodies.The reporter cells were incubated for 18 hours at 37° C. and 5% CO₂.After the incubation, luciferase expression was detected using Bio-Glo(Promega) and an EnVision multilabel reader 2100.

The anti-GITR antibodies tested in this assay were Hum231#2w and 20germline variants: pab1964, pab1965, pab1966, pab1967, pab1968, pab1969,pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977, pab1979,pab1980, pab1981, pab1983, pab2159, pab2160 and pab2161. As shown inFIGS. 30A-C, all the germline variants showed agonistic activity in theGITR NF-κB-luciferase reporter assay.

The germline variants were also tested for agonistic activity in theabsence of anti-CD3 antibody. On the day of the experiment, the GITRNF-κB-luciferase reporter cells (Promega) were resuspended in assaymedia (RPMI+1% FBS). The cells (100,000 cells per well) were added to a96-well plate that contained various concentrations (12.5, 10, 5, 2.5,1.25, and 0.625 μg/ml) of plate-bound anti-GITR antibodies. The reportercells were incubated for 6 hours at 37° C. and 5% CO₂. After theincubation, luciferase expression was detected using Bio-Glo (Promega)and an EnVision multilabel reader 2100.

The anti-GITR antibodies tested in this assay were m6C8, Hum231#2w and20 germline variants: pab1964, pab1965, pab1966, pab1967, pab1968,pab1969, pab1970, pab1971, pab1972, pab1973, pab1975, pab1976, pab1977,pab1979, pab1980, pab1981, pab1983, pab2159, pab2160 and pab2161. Allthe germline variants induced a dose dependent activation of thereporter cell line in the absence of an anti-CD3 antibody (FIGS. 30D-F).

6.6 Example 6: Epitope Characterization of Anti-GITR Antibodies

To characterize the epitope on human GITR to which the chimeric parental231-32-15 antibody and the humanized anti-GITR antibodies recognize,further studies were performed as described below.

6.6.1 Epitope Competition—Cell Binding Assay

To confirm that the humanized variant antibodies retained the epitopespecificity of the parental chimeric 231-32-15 antibody a cell bindingassay was performed. 1624-5 pre-B cells expressing the chimeric parental231-32-15 antibody were harvested and 1×10⁶ cells were resuspended in200 μl FACS buffer plus: i) biotinylated GITR (GITR-bio) (1:1000),preincubated for 15 min with 2 μg chimeric parental 231-32-15 antibody;ii) GITR-bio (1:1000), preincubated for 15 min with 2 μg Hum231#1; iii)GITR-bio (1:1000), preincubated for 15 min with 2 μg Hum231#2; or iv)GITR-bio (1:1000). The cells were incubated for 20 min at 4° C. and thenwashed with 4 ml FACS buffer and centrifuged for 5 min at 300 g at 4° C.The cell pellet was resuspended in 200 μl FACS buffer plusstreptavidin-PE (1:1000) and then incubated and washed as before. Thecells were then resuspended in 200 μl FACS buffer for analysis using aFACS-AriaII (BD Biosciences).

FIG. 31 shows that the humanized variant antibodies retained the epitopespecificity of the chimeric parental 231-32-15 antibody. The right-handprofile shows the binding of GITR-bio to 1624-5 pre-B cells expressingthe chimeric parental 231-32-25 antibody. However, when GITR-bio waspre-incubated with either chimeric parental 231-32-15, Hum231#1 orHum231#2 antibodies there was a loss of binding of GITR-bio to the1624-5 cells (left-hand profile). The overlapping FACS profiles indicatethat the humanized variants also show very similar GITR bindingproperties to each other and to the chimeric parental 231-32-15antibody.

6.6.2 Epitope Competition—Suspension Array Technology

Anti-GITR antibodies (25 μl) were diluted to 2 μg/ml in assay buffer(Roche 11112589001) and incubated with 1500 Luminex® beads (5 μl,Luminex Corp, no 5 LC10005-01) coupled with anti-human IgG(F(ab)₂-specific, JIR, 105-006-097) over night in 0.5 ml LoBind tubes(Eppendorf, 0030108.116) under shaking conditions, in the dark. Thismixture was then transferred to pre-wetted 96-well filter plates(Millipore, MABVN1250). Plates were washed twice with 200 μl/well PBS toremove unbound antibody. At the same time 20 μg/ml of either the sameanti-GITR antibodies, different anti-GITR antibodies, or assay bufferwere incubated with 20 μl (1 μg/ml) R-PE labeled GITR antigen (R&Dsystems, di-sulfide-linked homodimer; 689-GR; in-house labeled withAbDSerotec LYNX Kit, LNK022RPE) for 1 h in the dark at 650 rpm. The beadmixture and the antigen/antibody mixture were mixed 1:1 (20 μl fromeach) and incubated for one additional hour under shaking conditions(20° C., 650 rpm). Directly before the measurement 40 μl of assay bufferwas added to each well and analysis was performed using a Luminex® 200system (Millipore) and a readout of 100 beads in 48 μl sample volume.Binding was determined using the MFI values of the non-competed control(100% binding, only assay buffer as competing compound).

When the chimeric parental 231-32-15 antibody was used as the capturedantibody, full binding competition was observed with both humanizedvariants. When the anti-GITR antibody m6C8 was used as the capturedantibody, no competition of binding was observed with the chimericparental 231-32-15 antibody or the two humanized variants (data notshown). These results indicate that m6C8 and the anti-GITR antibodiesdescribed herein recognize different epitopes on human GITR.

6.6.3 Epitope Competition—Surface Plasmon Resonance

For epitope binning using surface plasmon resonance the “in tandemapproach” was used (Abdiche Y N et al., (2009) Analytical Biochemistry,386: 172-180). For that purpose different chip surfaces were generatedon a CM5 sensor chip (GE Healthcare, Series S CM5, BR-1005-30) usingimmobilization of different densities of GITR antigen (R&D systems,disulfide-linked homodimer; 689-GR). Flow cell 2 contained GITR antigenin low density (667 RU), medium density was assessed in flow cell 3(1595 RU) and in flow cell 4, high density was achieved (4371 RU). Inflow cell 1 ovalbumin (1289 RU, Pierce ThermoFisher 77120) wasimmobilized for reference. Immobilization was performed according to astandard protocol from the manufacturer (GE Healthcare) for aminecoupling (activation of surface with 0.4M EDC and 0.1M NHS, GEHealthcare Amine coupling kit, BR-1000-50). Unreacted groups wereinactivated with 1M ethanol-amine-HCl pH8.5. Afterwards anti-GITRantibodies were run through the different surfaces at a concentration of300 nM (45 μg/ml) for 240 s at 5 μl/min. Using these conditionssaturation of the GITR surface should have been reached. A dissociationtime of 60 s was included before adding the competing antibody (300 nM,5 μl/min). Regeneration of the chip surface was performed using 10 mMGlycine pH2.0 (GE Healthcare, BR-1003-55) for 60 s at 10 μl/min. Binningwas performed using the response units (RU) of the non-competed control(100% binding, saturating conditions).

As is shown in FIG. 32, when the chimeric parental 231-32-15 antibody isfirst bound to GITR no further binding of this antibody occurs. Howeverwhen the chimeric parental 231-32-15 antibody is first bound to GITR andthe antibody m6C8 is applied, this antibody is still able to bind toGITR.

6.6.4 Epitope Mapping of Anti-GITR Antibodies

In order to map the epitope on GITR to which anti-GITR antibodiesdescribed herein bind, error prone PCR was used to generate variants ofthe human GITR antigen. The variant GITR proteins were expressed on thesurface of cells in a cellular library and these cells were screened forbinding of the anti-GITR antibodies. As a positive control, a polyclonalanti-GITR antibody was used to confirm proper folding of the GITRprotein. For variants of the human GITR antigen to which reduced or noantibody binding occurred, alanine scanning mutagenesis was performed todetermine the precise epitope residues that were required for binding bythe anti-GITR antibodies described herein.

6.6.4.1 Generation of Human GITR Variants

Error prone PCR mutagenesis was used to generate variants of human GITRwith random mutations in the extracellular domain. For error prone PCR,the GeneMorphII Random Mutagenesis Kit (Stratagene) was used, accordingto the manufacturer's instructions. In brief, 20 PCR cycles in a volumeof 50 μl was performed using an in-house construct as template (13 ng,construct number 4377 pMA-T-huGITR), 0.05U/μl Mutazyme II DNApolymerase, 1× Mutazyme II reaction buffer, 0.2 μM of each primer(1152-Je (Sequence 5′ gagctcctcgaggccaccatg 3′; SEQ ID NO: 712) and1204-Je (Sequence 5′ cgcggccgcgaattctta 3′; SEQ ID NO: 713)) and 0.2 mMof each deoxynucleoside-triphosphate (dATP, dCTP, dGTP, and dTTP). Thesamples were amplified by PCR (Eppendorf, Germany) using the followingprogram: 95° C. for 2 min; 20 cycles of 95° C. for 30 sec, 56° C. for 30sec, 72° C. for 1 min; and a final extension step of 72° C. for 10 min.The PCR product was gel purified using 1% agarose gel, the DNA bandcorresponding to the expected size of 720 bp was cut out and gelextraction was done using a NucleoSpin Gel and PCR cleanup kit fromMacherey&Nagel according to the product manual. Purified DNA was ligatedinto an in-house expression vector via XhoI/EcoRI sites using T4 DNAligase and a ratio of 1:3 (vector:insert). Ligation (25° C.) was stoppedafter 2 hours with a heat denaturation step for 10 min at 65° C. DNAfrom the ligation reaction was EtOH precipitated using yeast t-RNA.Standard digestion and ligation techniques were used. The ligationreaction was electroporated into DH10B cells (E. coli ElectroMax DH10Belectrocompetent cells, Invitrogen; 1900V/5 ms). Electroporated bacteriawere plated onto LB-agar+100 μg/ml ampicillin plates and approximately1.9×10⁸ colonies were obtained.

All electroporated bacteria were then scratched from the plates and usedfor large-scale DNA plasmid preparation (Macherey&Nagel, NucleoBond XtraMaxi Plus Kit), according to the manufacturer's instructions to generatea DNA library. A restriction enzyme digestion with XhoI/EcoRI andBsrGI/EcoRI was performed to quality control the library. Single cloneswere picked and sent for sequencing to determine the final librarydiversity using primer 1155-Je (fwd; Sequence 5′ ccttgaacctcctcgttcg 3′;SEQ ID NO: 714).

6.6.4.2 Generation of a Cellular Library with Human GITR Variants

Standard techniques of transfection followed by transduction were usedto express human GITR mutants on the surface of 1624-5 cells. For thegeneration of retroviral particles, a DNA library and vectors expressingretroviral proteins Gag, Pol and Env were transfected into a retroviralpackaging cell line (HEK cells) using X-tremeGENE 9 DNA transfectionreagent (Roche Diagnostics GmbH, Germany). The resulting retroviralparticles accumulated in the cell culture supernatant of the retroviralpackaging cells. Two days post transfection cell-free viral vectorparticle-containing supernatants were harvested and subjected tospin-infection of 1624-5 cells. A transduction efficiency (% human GITRexpressing cells) of roughly 4% was obtained. Upon continuous culturefor at least one additional day, cells were selected using puromycin(1.5 μg/ml). Untransduced cells served as negative controls (NC). Afterantibiotic selection, most cells stably expressed the human GITR antigenlibrary on the cell surface. Non-viable cells were removed via a Ficollseparation step.

FACS was used to select cells expressing correctly folded human GITRmutants using a polyclonal anti-GITR antibody and to subsequently selectindividual cells expressing human GITR variants that did not bind to theanti-GITR chimeric parental 231-32-15 antibody. In brief, antibodybinding cells were analyzed by FACS and cells that exhibited specificantibody binding were separated from the non-binding cell population bypreparative, high-speed FACS (FACSAriaII, BD Biosciences). Antibodyreactive or non-reactive cell pools were expanded again in tissueculture and, due to the stable expression phenotype of retrovirallytransduced cells, cycles of antibody-directed cell sorting and tissueculture expansion were repeated, up to the point that a clearlydetectable anti-GITR antibody (chimeric parental 231-32-15) non-reactivecell population was obtained. This anti-GITR antibody (chimeric parental231-32-15) non-reactive cell population was subjected to a final,single-cell sorting step. After several days of cell expansion, singlecell sorted cells were again tested for non-binding to anti-GITRchimeric parental 231-32-15 antibody and binding to a polyclonalanti-GITR antibody using 96 well plate analysis on a FACSCalibur (BDBiosciences).

6.6.4.3 Epitope Analysis

To connect phenotype (polyclonal anti-GITR+, chimeric parental231-32-15-) with genotype, sequencing of single cell sorted huGITRvariants was performed. FIG. 33 shows the alignment of sequences fromthese variants. The amino acid residues in FIG. 33 are numberedaccording to the immature amino acid sequence of human GITR (SEQ ID NO:701). Sequencing identified regions with increased mutations or “hotspots” (e.g., P62 and G63), providing an indication of the epitope onhuman GITR recognized by anti-GITR chimeric parental 231-32-15 antibody.

To confirm the precise amino acids of human GITR involved in binding toanti-GITR antibodies, alanine replacement of hot spot amino acids wasperformed. The following positions (numbered according to SEQ ID NO:701) were separately mutated to an Alanine: P28A, T29A, G30A, G31A,P32A, T54A, T55A, R56A, C57A, C58A, R59A, D60A, Y61A, P62A, G63A, E64A,E65A, C66A, C67A, S68A, E69A, W70A, D71A, C72A, M73A, C74A, V75A, andQ76A. Standard techniques of transfection followed by transduction wereused to express these human GITR alanine mutants on the surface of1624-5 cells.

Finally, alanine mutants expressed on 1624-5 cells were tested in flowcytometry (FACSCalibur; BD Biosciences) for the binding of the anti-GITRhumanized variant Hum231#2, three germline variants (pab1967, pab1975and pab1979) and the reference antibody m6C8. Briefly, 1624-5 cellsexpressing individual human GITR alanine mutants were incubated with 2μg/ml of the monoclonal anti-GITR antibodies Hum231#2, three germlinevariants (pab1967, pab1975 and pab1979), or the m6C8 antibody; or apolyclonal anti-GITR antibody (AF689, R&D systems) conjugated with APC,and Fc receptor block (1:200; BD Cat no. 553142) diluted in 100 μl FACSbuffer (PBS+2% FCS) for 20 min at 4° C. After washing, the cells wereincubated with a secondary anti-IgG antibody if necessary for detection(APC conjugated; BD Cat no. 109-136-097) diluted in 100 μl FACS buffer(PBS+2% FCS) for 20 min at 4° C. The cells were then washed and acquiredusing a flow cytometer (BD Biosciences). The mean fluorescence intensity(MFI) value of the tested monoclonal antibody was divided by the MFIvalue of the polyclonal antibody, generating an MFI ratio (monoclonalantibody/polyclonal antibody) for individual GITR alanine mutants. Anaverage MFI ratio (“AMFI ratio”) was calculated based on the individualMFI ratios for all the mutants. FIG. 34A is a table summarizing thebinding of Hum231#2, three germline variants (pab1967, pab1975 andpab1979) and the reference antibody m6C8 to1624-5 cells expressing humanGITR alanine mutants. An individual MFI ratio that is above 60% of theAMFI ratio is considered to indicate similar binding, afternormalization, of that of the polyclonal antibody and is represented by“+” in FIG. 34A. An individual MFI ratio that is between 30% and 60% ofthe AMFI ratio is represented by “+/−” in FIG. 34A. An individual MFIratio that is below 30% of the AMFI ratio is represented by “−” in FIG.34A.

As shown in FIG. 34A, the D60A mutant and the G63A mutant, numberedaccording to SEQ ID NO: 701, specifically disrupted or weakened thebinding of the anti-GITR humanized variant Hum231#2 and the threegermline variants (pab1967, pab1975 and pab1979), but not that of thereference antibody m6C8. The C58A mutant disrupted the binding of allfive antibodies and is likely a structural mutation rather than anepitope-specific one. The C74A mutant had weak expression and could notbe used for binding comparison.

Furthermore, the anti-GITR antibodies 231-32-15, Hum231#2, and m6C8 werecompared for their binding to wild type versus mutant human GITR.Briefly, wild type human GITR and two GITR alanine mutants (the D60Amutant and the G63A mutant, numbered according to SEQ ID NO: 701) wereexpressed on the surface of 1624-5 cells as described above and testedin a flow cytometry analysis as described above where cells were firststained using 2 μg/ml of the monoclonal antibodies 231-32-15, Hum231#2,and m6C8, or a polyclonal antibody conjugated to APC, and then stainedusing a secondary anti-IgG antibody if necessary for detection (APCconjugated; 1:1000; BD Cat No. 109-136-097). All the mean fluorescenceintensity (MFI) values were calculated as the mean of two measurements.The MFI value of the tested monoclonal antibody for a particular celltype was divided by the MFI value of the polyclonal antibody for thesame cell type, generating a total of nine MFI ratios (monoclonalantibody/polyclonal antibody): MFI ratio_(231-32-15, WT), MFIratio_(Hum231#2, WT), MFI ratio_(m6C8, WT), MFI ratio_(231-32-15, D60A),MFI ratio_(Hum231#2, D60A), MFI ratio_(m6C8, D60A), MFIratio_(231-32-15, G63A), MFI ratio_(Hum231#2, G63A), and MFIratio_(m6C8, G63A). The percentage of binding of an antibody to the GITRalanine mutants relative to the wild type GITR was calculated bydividing a particular MFI ratio for the GITR alanine mutants by thecorresponding MFI ratio for the wild type (e.g., dividing MFIratio_(Hum231#2, D60A) by MFI ratio_(Hum231#2, WT)). The percentage ofreduction in binding was determined by calculating, e.g., 100%*(1−(MFIratio_(Hum231#2, D60A)/MFI ratio_(Hum231#2, WT))).

As shown in FIG. 34B, the D60A mutant and the G63A mutant specificallydisrupted or weakened the binding of 231-32-15 and Hum231#2, but notthat of m6C8. The percentages shown in FIG. 34B are the percentages ofGITR positive cells in each plot. When tested using the cells expressingGITR D60A, antibody binding was reduced by 82% and 88% for 231-32-15 andHum231#2, respectively, compared with a 10% reduction for m6C8.Similarly, when tested using the cells expressing GITR G63A, the bindingof 231-32-15 and Hum231#2 was reduced by 37% and 59%, respectively,whereas the binding of m6C8 was increased by 62%.

As further evidence for the binding characteristics of the anti-GITRantibodies, the binding of the antibodies to cynomolgus GITR wascompared. The immature protein of cynomolgus GITR comprises the aminoacid sequence of SEQ ID NO: 704. To increase protein expression, thefirst residue of the signal peptide of cynomolgus GITR was replaced bymethionine, generating VIM cynomolgus GITR. A mutant cynomolgus GITRV1M/Q62P/S63G, where the amino acid residues at the positions 62 and 63(GlnSer), numbered according to SEQ ID NO: 704, were replaced by thecorresponding residues in human GITR (ProGly), was then generated. FIG.35A is a sequence alignment between human GITR, V1M cynomolgus GITR, andV1M/Q62P/S63G cynomolgus GITR. The three proteins shown in FIG. 35A wereexpressed on the surface of 1624-5 cells as described above and testedin a flow cytometry analysis as described above where cells were firststained using 2 μg/ml of the monoclonal antibodies 231-32-15, Hum231#2,and m6C8, or a polyclonal antibody conjugated to APC, and then stainedusing a secondary anti-IgG antibody (APC conjugated; 1:1000; BD Cat no.109-136-097).

As shown in FIG. 35B, the anti-GITR antibodies 231-32-15 and Hum231#2displayed binding only to the cells expressing V1M/Q62P/S63G cynomolgusGITR, but not the cells expressing V1M cynomolgus GITR.

6.7 Example 7: Treatment of T Cells In Vitro with Agonistic Anti-GITRAntibodies Followed by T Cell Infusion

T cells expressing GITR, indicating an activated status, may be furtheractivated to kill target cells, such as tumor cells, if cultured with aGITR agonistic antibody, e.g., Hum231#1, Hum231#2, Hum231#2w, or anotherGITR agonistic antibody described herein, prior to infusion into asubject, e.g., a human subject having cancer.

The expression level of GITR on T cells isolated from a subject, e.g., ahuman subject, is assessed by standard techniques, e.g., FACS analysis.The source of T cells is, e.g., peripheral blood, a biopsy/surgicalspecimen of a lymph node that drains the tumor site, or abiopsy/surgical specimen of the tumor itself in which T cells may haveinfiltrated. The T cells are isolated, e.g., from total PBMCs, lymphtissue or tumor tissue by standard techniques. If GITR is observed to beexpressed on the T cell surface, the cells may be incubated with a GITRagonistic antibody, e.g., Hum231#1, Hum231#2, Hum231#2w, or another GITRagonist antibody described herein, at concentrations ranging from, e.g.,1 μg/ml to 1 mg/ml, for, e.g., 30 minutes, 1 hour, 2 hours, 3 hours, 4hours, 6, hours, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours,followed by, e.g., intravenous infusion of the T cells into a subject.

If GITR is not observed to be expressed on T cells isolated from asubject, its expression may be induced by co-incubation of the T cellswith a TCR complex stimulating agent, such as e.g., phytohaemagglutinin(PHA) and/or phorbol myristate acetate (PMA), or a TCR complexstimulating antibody, such as an anti-CD3 antibody and anti-CD28antibody, (at concentrations ranging from, e.g., 1 μg/ml to 1 mg/ml,for, e.g., 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6, hours, 12hours, 24 hours, 36 hours, 48 hours, or 72 hours). Alternatively, it maybe preferable to first incubate T cells with an anti-CD3 antibody alonefollowed by addition of the agonistic GITR antibody 30 mins, 1 hour, 2hours, 3 hours, 4 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48hours, or 72 hours later. It may also be desirable to stimulate T cellswith a tumor antigen, e.g., in the form of peptides or proteins, in thepresence of antigen presenting cells in order to activate and expand thenumber of tumor-specific T cells. Subsequent to antigen stimulation, theT cells may be cultured with a GITR agonistic antibody to enhance theiractivation status prior to infusion.

The T cells may be infused into the subject over a period of, e.g., 30minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6, hours, 12 hours, or 24hours, and may be infused into the subject one, two, three, four, ormore times, e.g., separated by 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, or6 months. The number of T cells infused may be determined by standardexperimental methods and may include, e.g., 1×10⁶ cells, 1×10⁷ cells,1×10⁸ cells, 1×10⁹ cells or more.

In some embodiments, the T cells may be contacted with an agent such asa mitogen (e.g., PHA) or a cytokine (e.g., IL-2) to non-specificallyexpand the T cell population prior to, during, or subsequent totreatment with GITR agonistic antibody.

In some embodiments, the T cells may be contacted with a further agonistin addition to the GITR agonistic antibody, e.g., an OX40 agonisticantibody.

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entirety andfor all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes. Other embodiments are within the following claims.

1-205. (canceled)
 206. An isolated antibody that specifically binds tohuman GITR, comprising: (a) a heavy chain variable region (VH)comprising a VH CDR1 comprising the amino acid sequence of DYAMY (SEQ IDNO: 13), a VH CDR2 comprising the amino acid sequence ofVIRTYSGDVTYNQKFKD (SEQ ID NO: 14), and a VH CDR3 comprising the aminoacid sequence of SGTVRGFAY (SEQ ID NO: 15); and (b) a light chainvariable region (VL) comprising a VL CDR1 comprising the amino acidsequence of KSSQSLLNSGNQKNYLT (SEQ ID NO: 16), a VL CDR2 comprising theamino acid sequence of WASTRES (SEQ ID NO: 17), and a VL CDR3 comprisingthe amino acid sequence of QNDYSYPYT (SEQ ID NO: 18).
 207. The antibodyof claim 206, wherein the VH comprises the amino acid sequence of SEQ IDNO:
 206. 208. The antibody of claim 206, wherein the VL comprises theamino acid sequence of SEQ ID NO:
 208. 209. The antibody of claim 206,wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 206;and (b) the VL comprises the amino acid sequence of SEQ ID NO:
 208. 210.The antibody of claim 209, wherein: (a) the amino acid sequence of theVH consists of the amino acid sequence of SEQ ID NO: 206; and (b) theamino acid sequence of the VL consists of the amino acid sequence of SEQID NO:
 208. 211. The antibody of claim 209, wherein the antibody furthercomprises a human immunoglobulin IgG₁ heavy chain constant region. 212.The antibody of claim 211, wherein the antibody further comprises ahuman immunoglobulin IgG kappa light chain constant region.
 213. Theantibody of claim 209, wherein the antibody further comprises a humanimmunoglobulin IgG kappa light chain constant region.
 214. The antibodyof claim 206, wherein the antibody comprises a heavy chain comprisingthe amino acid sequence of SEQ ID NO:
 567. 215. The antibody of claim214, wherein the VL comprises the amino acid sequence of SEQ ID NO: 208.216. The antibody of claim 206, wherein the antibody comprises a lightchain comprising the amino acid sequence of SEQ ID NO:
 576. 217. Theantibody of claim 216, wherein the VH comprises the amino acid sequenceof SEQ ID NO:
 206. 218. The antibody of claim 206, wherein the antibodyis produced by culturing a host cell comprising a nucleic acid moleculeencoding the amino acid sequence of SEQ ID NO: 206 so that the nucleicacid molecule is expressed and the antibody is produced.
 219. Theantibody of claim 218, wherein the host cell further comprises a nucleicacid molecule encoding the amino acid sequence of SEQ ID NO:
 576. 220.The antibody of claim 206, wherein the antibody is produced by culturinga host cell comprising a nucleic acid molecule encoding the amino acidsequence of SEQ ID NO: 576 so that the nucleic acid molecule isexpressed and the antibody is produced.
 221. An isolated antibody thatspecifically binds to human GITR, wherein the antibody is produced byculturing a host cell comprising a first nucleic acid molecule encodingthe amino acid sequence of SEQ ID NO: 206 and a second nucleic acidmolecule encoding the amino acid sequence of SEQ ID NO: 208 so that thefirst nucleic acid molecule and the second nucleic acid molecule areexpressed and the antibody is produced.
 222. The antibody of claim 221,wherein the host cell is a CHO cell.
 223. The antibody of claim 222,wherein the first nucleic acid molecule and the second nucleic acidmolecule are in a single vector.
 224. The antibody of claim 222, whereinthe first nucleic acid molecule is in a first vector and the secondnucleic acid molecule is in a second vector.
 225. An isolated antibodythat specifically binds to human GITR, comprising: (a) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 567; and (b) a lightchain comprising the amino acid sequence of SEQ ID NO:
 576. 226. Theantibody of claim 225, wherein: (a) the amino acid sequence of the heavychain consists of the amino acid sequence of SEQ ID NO: 567; and (b) theamino acid sequence of the light chain consists of the amino acidsequence of SEQ ID NO:
 576. 227. A pharmaceutical compositioncomprising: an isolated antibody that specifically binds to human GITR,comprising: (a) a heavy chain variable region (VH) comprising a VH CDR1comprising the amino acid sequence of DYAMY (SEQ ID NO: 13), a VH CDR2comprising the amino acid sequence of VIRTYSGDVTYNQKFKD (SEQ ID NO: 14),and a VH CDR3 comprising the amino acid sequence of SGTVRGFAY (SEQ IDNO: 15); and (b) a light chain variable region (VL) comprising a VL CDR1comprising the amino acid sequence of KSSQSLLNSGNQKNYLT (SEQ ID NO: 16),a VL CDR2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 17),and a VL CDR3 comprising the amino acid sequence of QNDYSYPYT (SEQ IDNO: 18); and a pharmaceutically acceptable carrier or excipient. 228.The pharmaceutical composition of claim 227, wherein: (a) the VHcomprises the amino acid sequence of SEQ ID NO: 206; and (b) the VLcomprises the amino acid sequence of SEQ ID NO:
 208. 229. Thepharmaceutical composition of claim 228, wherein the antibody furthercomprises a human immunoglobulin IgG₁ heavy chain constant region. 230.The pharmaceutical composition of claim 229, wherein the antibodyfurther comprises a human immunoglobulin IgG kappa light chain constantregion.
 231. The pharmaceutical composition of claim 228, wherein theantibody further comprises a human immunoglobulin IgG kappa light chainconstant region.
 232. The pharmaceutical composition of claim 227,wherein: (a) the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 567; and (b) the VL comprises the amino acidsequence of SEQ ID NO:
 208. 233. The pharmaceutical composition of claim227, wherein: (a) the antibody comprises a light chain comprising theamino acid sequence of SEQ ID NO: 576; and (b) the VH comprises theamino acid sequence of SEQ ID NO:
 206. 234. The pharmaceuticalcomposition of claim 227, wherein the antibody is produced by culturinga host cell comprising a first nucleic acid molecule encoding the aminoacid sequence of SEQ ID NO: 206 and a second nucleic acid moleculeencoding the amino acid sequence of SEQ ID NO: 208 so that the firstnucleic acid molecule and the second nucleic acid molecule are expressedand the antibody is produced.
 235. The pharmaceutical composition ofclaim 234, wherein the host cell is a CHO cell.